The current study demonstrates that the sympathetic neural recruitment patterns observed during chemoreflex activation induced by rebreathing or apnea are restrained and/or inhibited by the act of ventilation per se, despite similar, or even greater, levels of severe chemoreflex stress. Therefore, ventilation modulates not only the timing of sympathetic bursts but also the within-burst axonal recruitment normally observed during progressive chemoreflex stress. 1) functional residual capacity apnea (FRCApnea) and 2) continued RBR. Data from RBR were analyzed at matched  hemoglobin saturation (HbSat) levels (RBRMatched) or more severe levels (RBREnd). A third protocol compared alternating periods (30 s) of FRC and RBR (FRC-RBRALT). Subjects continued each protocol until 85% volitional tolerance. AP patterns in MSNA  were studied using wavelet-based methodology. First, for similar levels of chemoreflex stress , RBRMatched was associated with reduced AP frequency and APs per burst compared with FRCApnea (both P < 0.001). When APs were binned according to peak-to-peak amplitude , total AP clusters increased during FRCApnea  but not during RBRMatched . Second, despite more severe chemoreflex stress during RBREnd , RBREnd was associated with a restrained increase in the APs per burst  and total AP clusters  (both P < 0.01). During FRC-RBRALT, all periods of FRC elicited sympathetic AP recruitment , whereas all periods of RBR were associated with complete withdrawal of AP recruitment . Presently, we demonstrate that ventilation per se restrains and/or inhibits sympathetic axonal recruitment during high, and even extreme, chemoreflex stress.This study investigated the influence of ventilation on sympathetic action potential (AP) discharge patterns during varying levels of high chemoreflex stress. In seven trained breath-hold divers (age 33 ± 12 yr), we measured muscle sympathetic nerve activity (MSNA) at baseline, during preparatory rebreathing (RBR), and during NEW & NOTEWORTHY The current study demonstrates that the sympathetic neural recruitment patterns observed during chemoreflex activation induced by rebreathing or apnea are restrained and/or inhibited by the act of ventilation per se, despite similar, or even greater, levels of severe chemoreflex stress. Therefore, ventilation modulates not only the timing of sympathetic bursts but also the within-burst axonal recruitment normally observed during progressive chemoreflex stress. As such, the homeostatic challenges presented by central and/or peripheral chemoreceptor stimulation are corrected through reflexive increases in ventilation, as well as autonomic circulatory arousal  vs. the chemoreflex activation itself. For example, voluntary apnea potentiated the sympathetic response to isocapnic hypoxia, indicating the sympathoinhibitory nature of ventilation , 1989b. Much remains uncertain regarding the interactions between chemoreflex stress and ventilation on sympathetic neurocirculatory control. For example, the degree of chemoreflex stress may be an important caveat in understanding the role of respiratory movements in sympathetic control. The central motor hypothesis has been studied in humans mainly under normoxic and normocapnic conditions  or low lIn addition, the mode of studying sympathetic nerve activity likely has an impact on determinations of chemoreflex vs. ventilatory control over sympathetic nerve recruitment. Indeed, early work from single-unit action potential (AP) recordings demonstrated that the sympathetic adjustment to apnea involves not only an increase in neural firing probability but also a shift toward greater within-burst firing of single axons . TherefoTherefore, the objective of the current investigation was to determine the influence of ventilation on sympathetic AP recruitment during varying levels of high chemoreflex stress. To do so, we studied sympathetic AP discharge during apnea at FRC  and during asphyxic rebreathing  in trained breath-hold divers. Trained breath-hold divers were studied because of their unique ability to perform apneas for prolonged periods of time and their capacity to endure high levels of chemoreflex stress . We test2) participated in the current investigation after providing informed written consent. Participants were nonsmokers without any history of cardiovascular or respiratory disease. All studies were conducted at the University of Split, adhering to the standards set by the latest revision of the Declaration of Helsinki, and all experimental procedures were approved by the Research Ethics Board at the University of Split School of Medicine in Croatia.Seven healthy individuals  and carbon dioxide (Pco2) partial pressures. End-tidal values for Po2  connected to a three-way valve, allowing them to breathe either room air, or through a Y-connector  leading to two 3-liter breathing bags. An infrared probe connected to a pulse oximeter  was applied to the index finger to monitor arterial oxygen saturation . Respiratory gases were analyzed using an infrared carbon dioxide sensor and optical oxygen detector fed from a damped micro-vacuum sampling pump  and calibrated using ambient air pressure values, which were converted to online measurements of oxygen  was completed in which baseline data were collected for 2 min, followed by alternating 30-s periods of FRC apnea and rebreathing until ~85% maximal tolerance.Participants completed three protocols in random order, each separated by 5 min of recovery. Before the onset of each protocol, participants expired into the breathing bags to fill them with air for an ensuing rebreathing period. The first protocol  assessed the impact of high levels of chemoreflex stress on sympathetic axonal recruitment in the absence of ventilation. Specifically, following 3 min of baseline data collection, the three-way valve was turned at end-expiration to begin an initial period of rebreathing, in an attempt to maximize the severity of chemoreflex stimulation incurred during the ensuing apnea period . This inMultiunit recordings of MSNA were obtained from the right peroneal nerve by microneurography  model 6. Heart rApnea). For the rebreathe protocol, data were analyzed for the 3-min baseline period, for the initial rebreathe period . Therefore, this represents the FRCApnea vs. RBRMatched comparison at the same level of chemoreflex stress, as inferred from the HbSat values .As a first aim, the study addressed the effect of the “absence or presence” of ventilation on sympathetic neural recruitment during similar  levels of chemoreflex stress. To address this aim, data were analyzed from the apnea protocol for the 3-min baseline period, the rebreathe period . The duration of data studied in the RBREnd period was matched to that of the last half of the FRCApnea, used in the first aim. Therefore, this represents the FRCApnea vs. RBREnd comparison.The second aim determined the influence of ventilation on sympathetic neural recruitment at more severe levels of chemoreflex stress than that induced by FRCALT protocol, because the number of alternating FRC apnea and rebreathe periods differed between participants, the 2-min baseline and the final three FRC apnea and rebreathe periods for each participant were used for analysis.A third aim addressed the issue that the apnea or continued rebreathe protocols always occurred at the end of each protocol, and therefore, we compared alternating periods of FRC apnea with rebreathe during progressively increasing chemoreflex stress. For the FRC-RBRMSNA data were analyzed in two manners. First, the integrated neurogram was quantified as per traditional methodology. Second, we studied the patterns of change in APs from the filtered raw MSNA signal as per our approach detailed previously . IntegraApnea vs. RBRMatched vs. RBREnd) protocols to ensure that bin width, maximum bin center, and the total number of bins would be identical across conditions. This procedure ensured that corresponding clusters within and between protocols contain APs with the same peak-to-peak amplitude. Therefore, within protocols , an increase in the number of total clusters represents recruitment of latent subpopulations of larger sized axons not present at baseline, whereas between protocols , a greater number of total clusters represents further recruitment of these latent subpopulations compared with other protocols. Sympathetic AP patterns were quantified using AP frequency (the number of APs per minute), whereas sympathetic AP recruitment was expressed using the mean AP content per integrated sympathetic burst (APs/burst), the number of active AP clusters per integrated sympathetic burst (clusters/burst), and the number of total AP clusters . Based on previous validation analysis of our technique  vs. time . For the FRC-RBRALT protocol, one-way repeated-measures ANOVA was used to assess the effect of time . Bonferroni-corrected post hoc procedures were used to assess specific differences between means, when appropriate. Statistical significance was set at P < 0.05, and all data are means ± SD.All statistical analyses were performed using SigmaPlot 12.0 . All data were normally distributed as confirmed by Shapiro-Wilk tests. Two-way repeated-measures ANOVA was used to assess the effect of protocol  at end-maneuver. By design, HbSat levels were similar  between maneuvers . Compared with baseline, Apnea: +17 ± 2 Torr; RBRMatched: +19 ± 2 Torr; both P < 0.001) increased, whereas Apnea: −68 ± 7 Torr; RBRMatched: −84 ± 7 Torr; both P < 0.001) decreased, during FRCApnea and RBRMatched.Both FRCApnea and RBRMatched protocols are outlined in Apnea: +29 ± 7 bursts/min; RBRMatched: +12 ± 9 bursts/min; both P < 0.01) and burst incidence  increased during both maneuvers; however, values of burst frequency and burst incidence were greater during FRCApnea than during RBRMatched (both P < 0.001). Similarly, burst amplitude  increased above baseline levels during both maneuvers, although levels were greater during FRCApnea (P < 0.001). As such, compared with baseline, total MSNA was elevated during both maneuver periods , yet, once again, levels of total MSNA were greater during FRCApnea (P < 0.001).Integrated MSNA indexes during the baseline, rebreathe, and maneuver periods of the FRCApnea and RBRMatched protocols. Specifically, compared with baseline, AP frequency  increased during both maneuvers; however, AP frequency was greater during FRCApnea compared with RBRMatched . This increase in AP frequency was due to the elevated levels of burst frequency but also to an increase in the AP content per integrated sympathetic burst , during both maneuvers, compared with baseline. The AP content per integrated sympathetic burst, however, was greater during FRCApnea than during RBRMatched . Furthermore, when APs were binned according to peak-to-peak amplitude , the number of total AP clusters increased during FRCApnea  but not during RBRMatched . As such, the number of total AP clusters was greater during FRCApnea (P < 0.001). Finally, the number of active clusters per burst  was elevated above baseline levels during both maneuvers, although levels were greater during FRCApnea than during RBRMatched (P < 0.01).Apnea: +18 ± 9 mmHg; RBRMatched: +10 ± 7 mmHg; both P < 0.01) increased during both maneuvers, yet the pressor response was greater during FRCApnea (P < 0.001). The greater pressor response was associated with a greater TPR during FRCApnea (P < 0.001). HR decreased during FRCApnea and increased during RBRMatched (both P < 0.05), whereas reductions in SV were similar between maneuvers (P = NS). Subsequently, compared with baseline, CO decreased during FRCApnea (P < 0.001) but remained unchanged during RBRMatched (P = NS).The hemodynamic responses to both maneuvers were consistent with the sympathetic response patterns as shown in End), the RBREnd maneuver elicited a larger reduction in HbSat vs. FRCApnea  and larger decreases in Apnea: −68 ± 7 Torr; RBREnd: −94 ± 4 Torr; both P < 0.001) were observed during RBREnd compared with FRCApnea (P < 0.001).By design, when rebreathing was allowed to continue and analyzed at the end  increased to similar levels during both maneuvers (P = NS), whereas burst incidence  increased to a greater extent during FRCApnea than during RBREnd (P = 0.001). Burst amplitude  increased above baseline levels during both maneuvers, although levels were greater during FRCApnea than during RBREnd (P < 0.05). Finally, compared with baseline, total MSNA was elevated during both maneuver periods ; however, levels of total MSNA were not different during FRCApnea and RBREnd (P = NS).Apnea and RBREnd protocols are displayed in Apnea: +1122 ± 534 APs/min; RBREnd: +912 ± 429 APs/min; P < 0.001) increased during both maneuvers; however, AP frequency was not different during FRCApnea and RBREnd . Once again, the increased AP frequency was due to the elevated burst frequency but also to an increase in the AP content per integrated sympathetic burst  during both maneuvers, compared with baseline. However, the AP content per integrated sympathetic burst was greater during FRCApnea than during RBREnd . Furthermore, when APs were binned according to peak-to-peak amplitude , the number of total AP clusters increased during FRCApnea  and RBREnd , yet total AP clusters was greater during FRCApnea (P < 0.01). Finally, the number of active clusters per burst  was elevated above baseline levels during both maneuvers, whereas no differences were observed during FRCApnea vs. RBREnd (P = NS).Sympathetic AP discharge indexes during the baseline, rebreathe, and maneuver periods of the FRCApnea and RBREnd are shown in Apnea: +18 ± 9 mmHg; RBRMatched: +20 ± 12 mmHg; both P < 0.001) increased similarly during both maneuvers (P = NS). However, this pressor response was achieved differently for FRCApnea and RBREnd. Specifically, compared with baseline, TPR increased during FRCApnea (P < 0.001) but not during RBREnd (P = NS), whereas CO increased during RBREnd (P < 0.01) but decreased during FRCApnea (P < 0.001). The latter was due to an increase in HR during RBREnd (P < 0.001) compared with no change during FRCApnea (P = NS) and a similar decrease in SV during both maneuvers (both P < 0.001).The hemodynamic responses to FRCALT protocol. HbSat levels were decreased compared with baseline during each of the final two FRC and RBR periods of the FRC-RBRalt protocol (P < 0.001). Compared with baseline, P < 0.001). No differences existed between corresponding periods of FRC and RBR for HbSat or P = NS).alt protocol. Specifically, compared with baseline, all integrated MSNA variables were elevated during each FRC , but not during any of the three RBR periods . As such, every corresponding FRC-RBR period was associated with greater levels of integrated MSNA during FRC compared with RBR .alt protocol are shown in P < 0.001), but not during any RBR period . Therefore, every corresponding FRC-RBR period was associated with greater AP recruitment during FRC than during RBR .Sympathetic AP discharge indexes at baseline and during the FRC and RBR periods of the FRC-RBR alt protocol are provided in P < 0.001), whereas TPR increased during each FRC , but not during RBR . HR was reduced during the final FRC compared with the final RBR (P < 0.001), whereas SV remained unchanged throughout the FRC-RBRalt protocol . CO decreased during each FRC (P < 0.001) and was above baseline levels during the first RBR period (P < 0.001), but not in each of the final two RBR periods (both P = NS).Finally, the hemodynamic responses to the FRC-RBRThe current study was designed to study the influences of chemoreceptor stimuli  and ventilation on sympathetic AP recruitment patterns during chemoreflex stress. First, we demonstrated that during similar levels of (high) chemoreflex stress, the act of ventilation per se restrained the increased firing of already active, lower threshold axons and, furthermore, inhibited the recruitment of latent, higher threshold axons that appeared during apnea. Second, recruitment of previously silent axons was observed during severe chemoreflex stimulation induced by the continued rebreathe protocol; however, sympathetic AP recruitment was still restrained compared with apnea. Finally, during alternating periods of apnea and rebreathe, the act of rebreathing between apneas was associated with a rapid and complete withdrawal of sympathetic AP recruitment, despite the ever-increasing chemoreflex stimulus. As such, it appears that the sympathoinhibitory effect of ventilation during chemoreceptor activation affects both the frequency of neuronal discharge and recruitment of latent axons, even at severe levels of chemoreflex stress.The sympathoexcitatory response to voluntary apnea is robust and includes a powerful stimulus for efferent sympathetic axonal recruitment . The mecMatched). Indeed, RBRMatched was associated with a lower firing frequency and a restrained increase in the number of APs per integrated burst such that, overall, sympathetic AP frequency increased ~700% during FRCApnea but only ~300% during RBRMatched. Furthermore, the capacity of the sympathetic nervous system to recruit latent neural subpopulations was inhibited during RBRMatched, despite the same chemoreflex load, indicating the profound effect of ventilation on AP recruitment patterns. Therefore, these data replicate earlier observations . Interestingly, in this scenario  the total integrated MSNA response was similar between protocols, but the underlying patterns of AP recruitment differed by maneuver. These data highlight a challenge associated with quantification of MSNA based on burst metrics.Nonetheless, the current data do support the idea that the sympathoinhibitory effect of ventilation is less effective at more severe levels of chemoreflex stress. Specifically, latent axons were recruited during the more severe chemoreflex stress induced by the continued rebreathe protocol, despite the presence of ventilation , sympathetic axonal recruitment did occur over time, albeit at levels that were less than those observed during apnea. These observations suggest that the sympathoinhibitory influence of breathing may be robust acutely but, perhaps, less effective over time even as the chemoreflex load continues to extreme levels.Therefore, the present data support previous findings that indicate ventilation per se is responsible for the sympathoinhibition across a wide range of chemoreflex stress. However, the potency of this inhibitory effect appears to depend on the temporal or situational conditions. For example, acute periods of rebreathe following apnea were associated with complete withdrawal of AP discharge. However, when ventilation continued against a background of progressive chemoreflex stress  and Natural Sciences and Engineering Research Council of Canada Grant 217916-2013 (J. K. Shoemaker). M. B. Badrov was supported by a Canadian Institutes of Health Research Doctoral Scholarship Award and a Canada Graduate Scholarships – Michael Smith Foreign Study Supplement Award. J. K. Shoemaker is a Tier 1 Canada Research Chair.No conflicts of interest, financial or otherwise, are declared by the authors.M.B.B., O.F.B., I.D., Z.D., and J.K.S. conceived and designed research; M.B.B., O.F.B., T.M., M.L., I.D., Z.D., and J.K.S. performed experiments; M.B.B., L.N.S., and L.J.B. analyzed data; M.B.B., Z.D., and J.K.S. interpreted results of experiments; M.B.B. and L.J.B. prepared figures; M.B.B. drafted manuscript; M.B.B., O.F.B., T.M., L.N.S., L.J.B., Z.D., and J.K.S. edited and revised manuscript; M.B.B., O.F.B., T.M., L.N.S., L.J.B., M.L., I.D., Z.D., and J.K.S. approved final version of manuscript.


Transfluthrin vapour prevents mosquito bites by disrupting their host-seeking behaviors. We measured the additional benefits of combining transfluthrin-treated sisal decorations and long-lasting insecticidal nets (LLINs) with an aim of extending protection against early evening, indoor-biting malaria vectors when LLINs are ineffective.2) treated with 2.5 ml and 5.0 ml transfluthrin, in terms of mosquito density, exposure to bites and 24 h mortality. Experiments were conducted in experimental huts, located in Lupiro village, Ulanga District, south-eastern Tanzania. Human landing catches (HLC) were used to measure exposure to bites between 19:00–23:00 h. Each morning, at 06:00 h, mosquitoes were collected inside huts and in exit traps and monitored for 24 h mortality.We investigated the indoor protective efficacy of locally made sisal decorative baskets (0.28 m2) treated with 2.5 ml and 5.0 ml transfluthrin deterred three-quarters of Anopheles arabiensis mosquitoes from entering huts . Both treatments induced a 10-fold increase in 24 h mortality of An. arabiensis mosquitoes .Sisal decorative baskets (0.28 mSisal decorative items treated with spatial repellents provide additional household and personal protection against indoor biting malaria and nuisance mosquitoes in the early evening, when conventional indoor vector control tools, such as LLINs, are not in use. We recommend future studies to investigate the epidemiological relevance of combining LLINs and transfluthrin decorated baskets in terms of their effect on reduction in malaria prevalence. Long-lasting insecticidal nets (LLINs), indoor residual spraying (IRS), improved diagnosis and treatment have brought about substantial decline in malaria transmission, particularly in sub-Saharan Africa –3. DespiEffectiveness of LLINs depends on factors that influence human-vector contact, such as time and place of malaria-transmitting mosquito bites , user’s Topical repellents , 12 and Spatial repellents are vapour-phase insecticides that incapacitate mosquitoes and prevent them from locating hosts and obtaining blood meals . ExamplePrevious studies have shown that transfluthrin prevents mosquitoes from feeding , and indAn. arabiensis and Anopheles funestus (sensu lato) [An. arabiensis and An. funestus were pyrethroid  resistant [An. funestus (s.l.) was also resistant to pyrethroids (i.e. permethrin: 10.5%) [The study was conducted in Lupiro village , Ulanga District, south-eastern Tanzania . Annual su lato) . The maisu lato) , 24. A msu lato) . Precediesistant  and find: 10.5%) .2 were treated with either 2.5 ml or 5 ml of 97% transfluthrin  following the method previously described [Circular pieces of sisal 0.28 mescribed . Controlescribed . All pieescribed .Sisal fabrics are versatile products from the sisal plant, available in most of the tropical countries like Tanzania. These fabrics can be made into various household products, such as mats, baskets, curtains, wall picture frame, etc. The uniqueness of the sisal fabrics are: (i) they have relatively high absorbance of liquid such as water; and (ii) they allow slow release of transfluthrin in air, this way transfluthrin-treated sisal fabrics may remain effective for a duration of more than six months or a year , 31. NevAn. gambiae (s.l.) and An. funestus (s.l.) mosquitoes that were randomly selected each day. The primary outcomes measured included: (i) mosquito deterrence, which is reduction in the density of indoor mosquitoes; (ii) indoor human mosquito biting rate, which is the proportion of mosquitoes that landed and attempted to bite volunteers that were conducting HLC; and (iii) insecticide-induced 24 h mortality.Experiments were conducted from 6th January 2015 to 7th February 2015. The effect of combining transfluthrin-treated sisal baskets and permethrin-treated LLINs on the proportion of indoor mosquito density, the proportion of early evening indoor mosquito bites and survival of mosquitoes in experimental huts Fig.  was invelme4 package [An. arabiensis, An. funestus (s.l.) and Culex species mosquitoes were analyzed in separate models. The same analysis was used to measure reduction in the proportion of biting mosquitoes in the early evening. The total number of mosquitoes collected by HLC in experimental huts between 19:00 and 23:00 h was fitted as the dependent variable. The hut and the day of experiment were treated as random variables, while treatment arm was coded as a fixed variable. Insecticide induced mortality was determined by fitting a GLMMs with a binomial distribution and a logit-link function. The proportion of dead and live mosquitoes was coded as dependent binomial variable, treatment arms as fixed variable whereas day of experiment and experimental huts were treated as random variables.Deterrence was determined statistically using log-normal Poisson generalized linear mixed effects models (GLMMs) in R statistical software version 3.1.3, with  package . The resCulex spp.; 1672 An. arabiensis; 1165 An. funestus (s.l.); 121 Mansonia spp.; 4 Coquilettidia spp.; 3 An. coustani; and 3 Aedes spp. Of 91 An. gambiae (s.l.) samples amplified by PCR, all of the 86% (n = 78) successful amplifications achieved were An. arabiensis. Sixty-eight An. funestus (s.l.) samples were analyzed by PCR, and 71% (49/68) were successful amplifications. Of the successful amplifications, 96% (47/49) were An. funestus (sensu stricto), 2% (1/49) were An. leesoni, and the remaining 2% (1/49) were An. rivulorum.The total number of mosquitoes collected was 7125. These included 4157 An. arabiensis mosquitoes  and  (Table An. funestus (s.l.) mosquitoes . Huts with transfluthrin-treated sisal baskets and LLINs had nearly one third less Culex sp. mosquitoes compared to those with LLINs and untreated baskets  for 2.5 ml transfluthrin and  for 5 ml transfluthrin. As shown in Fig. Relative to LLINs with untreated sisal baskets, sisal decorative baskets treated with 2.5 ml and 5.0 ml transfluthrin, in combination with permethrin LLINs, reduced almost three quarters of indoor An. arabiensis mosquito bites by more than three quarters  compared to LLINs with untreated baskets. In addition, the two interventions reduced An. funestus (s.l.) mosquitoes bites by nearly half . The addition of transfluthrin-treated baskets reduced exposure to Culex spp. mosquitoes by approximately two thirds . Furthermore, as shown in Fig. Figure An. arabiensis . Compared to the control arm, adding transfluthrin-treated sisal baskets in experimental hut with LLINs, did not have impact on inducing mortality of An. funestus (s.l.) mosquitoes 24 h post-exposure . Neither 2.5 ml  nor 5.0 ml  transfluthrin-treated baskets combined with LLINs increased mortality of Culex spp. mosquitoes  in the presence of transfluthrin coils. However, we did not observe any added benefits of combining transfluthrin decorated baskets with LLINs in terms of inducing mortality of An. funestus and Culex spp. The low mortality observed for An. funestus may be partly explained by pyrethroid resistance exhibited by these mosquitoes as demonstrated previously [Mathematical models applied in previous studies postulate that a combination of repellent and LLINs attenuate community-wise benefit by diverting the vectors away from lethal, insecticide treated surfaces . SurprisNs Table , Fig. 4.eviously , and coneviously . The finAn. funestus (s.l.). This calls for frequent insecticide susceptibility tests to monitor emergence of resistance.Combining transfluthrin-treated household decorations and permethrin-treated LLINs was beneficial, and potentially enhanced protection by LLINs, against indoor biting malaria vectors by reducing indoor mosquito density and biting rate and increasing 24 h mortality. Transfluthrin is a pyrethroid, and its efficacy was less pronounced on suspected pyrethroid resistant Here, we have demonstrated that transfluthrin-treated emanators combined with LLINs reduce indoor mosquito entry and protect people against indoor mosquito bites when LLINs are not in effect. The emanators increase mortality of major malaria vectors in the area. Future studies should focus on measuring epidemiological endpoints of these combined interventions.


Neritopsis gedrosiana sp. nov., Calliostoma irerense sp. nov., Calliostoma mohtatae sp. nov. and Trivellona makranica sp. nov. are described as new species.A new gastropod fauna of Burdigalian (early Miocene) age is described from the Iranian part of Makran. The fauna comprises 19 species and represents three distinct assemblages from turbid water coral reef, shallow subtidal soft-bottom and mangrove-fringed mudflat environments in the northern Indian Ocean. Especially the reef-associated assemblage comprises largely new species. This is explained by the rare occurrence of reefs along the northern margin of the Miocene Indian Ocean and the low number of scientific studies dealing with the region. In terms of paleobiogeography, the fauna corresponds well to coeval faunas from the Pakistani Balochistan and Sindh provinces and the Indian Kathiawar, Kutch and Kerala provinces. During the early Miocene, these constituted a discrete biogeographic unit, the Western Indian Province, which documents the near complete biogeographic isolation from the Proto-Mediterranean Sea. Some mudflat taxa might represent examples of vicariance following the Tethys closure. The fauna also displays little connection with coeval faunas from Indonesia, documenting a strong provincialism within the Indo-West Pacific Region during early Miocene times.  The knowledge on the evolution and composition of the Miocene mollusc fauna of the Indian Ocean is still spotty due to the very limited fossil record. During the last years, Miocene gastropod assemblages from the Sultanate of Oman, coastal Tanzania, Kutch (NW-India) and Kerala (SW-India) have been described and revised by Harzhauser , 2014 anFirst descriptions of the faunas of the Balochistan and Sindh provinces and the Indian Kathiawar and Kutch provinces in Gujarat date back to the 19th century and start with the contributions by Sowerby  and ArchWhilst the mollusc faunas from the Pakistani part of the Makran were included by Vredenburg , 1928 thDitrupa coquinas. The latter contain some shells of potamidids and small turritellids. Wave ripples are commonly preserved at the top of thin sandstone beds. Intercalated within this alternation are several deposits of dark grey marl with abundant plant debris, in situ rootlets, Terebralia and certain septarian levels. A bed of pedogenic carbonate and a stromatolith were also found in contact to such a phytoclastic marl at the base of the investigated lithological succession. This facies association points to extensive tidal flats, which have been colonized by mangroves in the upper part. The next sedimentary unit is 11 m thick and consists of marls, calcareous marls and argillaceous limestones, which formed in shallow subtidal environments. It is conformably overlain by marly coral limestones (13 m thick) with characteristic sheet and platestone growth fabrics .Gastropoda Cuvier, Class Neritimorpha Golikov and Starobogatov, Subclass Cycloneritimorpha Bandel and Frýda, Order Neritopsoidea Gray, Superfamily Neritopsidae Gray, Family Neritopsis Grateloup Genus Type species. Neritopsis moniliformis Grateloup Neritopsis gedrosiana Harzhauser sp. nov.Figure Etymology. Referring to the ancient name Gedrosia for the Makran area.Holotype. NMHW 2017/0033/0001, height: 26.1 mm, diameter: 29.2 mm : diameter: 36 mm.Locality and horizon. Close to Irer village , Makran, Islamic Republic of Iran; reefal limestone of the Band-e-Chaker Formation, Burdigalian, early Miocene.Diagnosis. Large Neritopsis with densely spaced spiral rows of densely spaced, delicate beads separated by faint secondary spiral rows of beads along periphery.Description. Large for genus, wider than high, with weakly protruding spire consisting of c. two convex whorls; protoconch unknown. Last whorl large, rapidly widening, regularly convex at periphery, with narrow sutural ramp and distinct suture. Sculpture consisting of c. 30–35 densely spaced spiral rows of densely and regularly spaced rounded beads, which do not form an axial pattern. Spiral rows separated by narrow spiral grooves on adapical part of whorl but widening along periphery. Weak secondary spiral cords with faint beads may be intercalated between primary cords along periphery. Aperture subcircular, obscured by sediment; outer lip strongly prosocline, thin. Umbilicus concealed, indicated by shallow depression.Remarks. No Neritopsis species has been described so far from the Miocene of the Indian Ocean. The coeval Western Tethyan/Proto-Mediterranean Neritopsis moniliformis Grateloup Neritopsis radula —Ladd: p. 42, pl. 5, Figs. 29–34.cf. 1966 Angaria (Angaria) delphinus .Measurements. height: 33.0 mm, diameter: 35.1 mm.Description. Solid shell with broad, gradate spire comprising about five teleoconch whorls. Early spire whorls strongly abraded; last spire whorl angulated, with nearly flat sutural ramp. Sculpture consisting of broad, knob-like swellings close below upper suture and prominent triangular nodes along shoulder. Last whorl sloping in lower direction resulting in oblique suture. Low angled sutural ramp with four spiral rows of irregular blunt nodes; shoulder with wide spaced spiny nodes, pointing slightly in apical direction. Periphery flat, weakly contracting; transition into convex base angulated. Base covered by several spiral cords with broad nodes, which become scaly in the circum-umbilical area; umbilicus covered by sediment.Remarks. The sculpture of the specimen is partly abraded, which causes uncertainties about the strengths and lengths of the various nodes. The overall morphology agrees well with Angaria delphinus, which is widespread in the IWP-Neogene.Stratigraphic and geographic distribution. Fossils identified as Angaria delphinus are recorded from the lower Miocene of Gujarat and Kerala in India  petholatus Linnaeus – Ladd: p. 47, pl. 7, Figs. 21–22.cf. 1966 Turbo petholatus Linnaeus, cf. 2008 Material. Three specimens (NMHW 2017/0033/0014).Measurements. height: 29.5 mm, diameter: 44.1 mm; height: 37 mm (compressed): diameter: 50.5 mm.Description. Medium-sized turbinid with moderately high spire; spire whorls convex with faint shoulder; last whorl and base regularly convex, rapidly increasing in diameter. Shell surface smooth aside from prosocline growth lines and regularly spaced prosocline grooves, most prominent below suture and fading out below periphery. Weak spiral cord along upper suture. Umbilicus covered, indicated by shallow depression. Aperture circular with thickened and slightly reflected basal peristome.Remarks. The specimens are highly reminiscent of the extant Turbo petholatus. The small size and the deeper umbilical depression might indicate that the Iranian Burdigalian shells represent a separate species but the poor preservation does not allow a clear identification.Stratigraphic and geographic distribution. The earliest record of Turbo petholatus is documented from the early Miocene of Fiji , Makran, Islamic Republic of Iran; reefal limestone of the Band-e-Chaker Formation, Burdigalian, early Miocene.Diagnosis. Large depressed trochiform shell flange-like periphery and sculpture of 10–11 densely spaced, beaded spiral cords; flat base and narrow, open umbilicus.Description. Large, broad, depressed trochiform, solid shell; early spire slightly coeloconoid, later low cyrtoconoid. Protoconch and early teleoconch whorls not preserved. Early spire whorls with shallow concavity in lower third causing a flange-like outline of periphery. Concavity disappears gradually during growth. Sculpture consisting of 10–11 densely spaced and delicately beaded spiral cords; beads differ slightly in strength on individual spiral cords and do not form any axial pattern. Interspaces between spiral cords smooth, very narrow. Suture weakly incised. Periphery of fully grown specimens weakly rounded passing into flat base, covered by c. 20 beaded spiral cords; umbilicus open, narrow.Remarks. This species is characterized by its broad trochiform outline and large size. No comparable species was described so far from the Miocene of the Indian Ocean. “Trochus” kathiawarensis Jain, Calliostoma selecta  and the smaller size and coeloconoid spire (C. benedicti). Its autochthonous occurrence within the reefal limestones, suggests that this species was associated with corals.Dillwyn, , from Neti Dall,  are someStratigraphic and geographic distribution. Only known from the type locality.Calliostoma mohtatae Harzhauser sp. nov.Figure Etymology. In honor of Tayebeh Mohtat .Holotype. NMHW 2017/0033/0009, height: 25.4 mm, diameter: 20.6 mm .Locality and horizon. Close to Irer village , Makran, Islamic Republic of Iran; reefal limestone of the Band-e-Chaker Formation, Burdigalian, early Miocene.Diagnosis. Medium-sized conical trochiform shell; early spire whorls with weak subsutural concavity and carina at lower suture. Sculpture consisting of six densely spaced and densely beaded spiral cords; flat base with beaded spiral cords, nearly closed umbilicus.Description. Medium-sized, conical trochiform shell of about 8–9 teleoconch whorls. Protoconch unknown. Early spire conical to slightly cyrtoconoid passing into conical teleoconch with an angle typically ranging around 40° (but attaining up to 50°). Early spire whorls always strongly abraded, bearing four delicately beaded spiral cords and a fifth prominent adsutural cord with protruding nodes, forming a carina at lower suture; carina restricted to one or two whorls; later teleoconch whorls covered by six densely spaced and densely beaded spiral cords; beads are often subquadratic on last two teleoconch whorls and especially in lower half of whorl. Upper half of whorl often coinciding with a shallow concavity; lower half often slightly bulgy with more prominent spiral cords. Two narrow, weakly beaded secondary spiral cords may be intercalated between lower three primary cords on last whorl. Periphery narrowly rounded passing into flat base with 11–13 beaded spiral cords. Beads on base are prominent close to periphery and become gradually weaker towards umbilicus. Umbilicus reduced to narrow chink, smooth, demarcated from base by rim-like spiral cord. Inner lip narrow, slightly thickened. Outer lip strongly prosocline, rapidly thinning, slightly flaring, with a single weak spiral ridge close to the base inside.Some specimens display remnants of color patterns consisting of small, vaguely axially arranged speckles on the base and broad, opisthocline axial bands on the spire whorls separated by slightly wider light bands.Remarks. The species is somewhat variable in shape ranging from slender to more bulky shells and in more or less bulgy outline of the lower half of the whorls. Calliostoma jujubiniforme , pl. 26 Fig. 3 . This species seems to represent a new species but the available specimens are too fragmentary to serve as types. Martin, , from th Martin,  used dolStratigraphic and geographic distribution. Only known from the Irer section in Iran, where it was found within the coral reef unit.Turritellidae Lovén, Family Zaria Gray, Genus Type species. Turbo duplicatus Linnaeus, Zaria angulata , pl. 26 Fig. 7.*1840 Turritella angulata Sowerby—Archiac and Haime: p. 294, pl. 27 Figs. 6–9.1853 Zaria angulata .Measurements. maximum diameter: 12.6 mm.Remarks. Due to the fragmentary preservation, the identification remains debatable. The wide apical angle and the very prominently keeled angulation, however, agree fully with Indian specimens described by Jain .Measurements. maximum diameter: 32.4 mm.Description. Medium-sized Tibia with weakly convex spire whorls and a moderately convex last whorl with slightly gradate transition into the spire. Inner lip strongly thickened, broad, well demarcated from base. Posterior canal short, narrow, with dorsally twisted tip, terminating distinctly below upper suture. Last whorl with weak shoulder close to the aperture.Remarks. Although the fragmentary preservation does not allow a clear identification, the strongly thickened callus of the inner lip and the short, twisted posterior canal are reminiscent of Tibia indica. A difference is the larger size of the Iranian shells and the slightly shorter posterior canal, which reaches up to the suture of the penultimate whorl. The late Miocene Tibia verbeeki , height: 17.5 mm, diameter: 11.3 mm.Locality and horizon. Close to Irer village , Makran, Islamic Republic of Iran; reefal limestone of the Band-e-Chaker Formation, Burdigalian, early Miocene.Diagnosis. Large elongate ovoid Trivellona with trapezoid profile and 12 × 2 prominent, rarely bifurcating dorsal ribs. Sharply edged labral shoulder coinciding with axial ridge forming beads at intersections with dorsal ribs. Central, nearly straight aperture with prominent denticles.Description. Shell elongate ovoid with subparallel margins. Dorsal profile trapezoid, moderately convex; dorsal sulcus indistinct, narrowly depressed. Sculpture consisting of 12  × 2 prominent, regular, subparallel and rarely bifurcating dorsal ribs , separated by broader interspaces; very weak secondary ribs are occasionally intercalated in the central part of the dorsum. Tips of primary ribs along dorsal sulcus only weakly out of phase in central part but slightly more so on terminal tips. Labral shoulder sharply edged coinciding with axial ridge forming beads at intersections with dorsal ribs. A second axial ridge occurs in the slightly angular ventral margin. Aperture in ventral center, very narrow, nearly straight. Labrum moderately convex to slightly flattened, declivous towards aperture throughout with 19 knobby labral denticles; parietal lip angularly edged with 17 columellar teeth projecting into aperture as prominent denticles. Posterior terminal tip blunt, anal canal short, narrow; anterior tip weakly protruding with short, narrow, twisted siphonal canal .cf. 2005 Material. One partial silicone cast (NMHW 2017/0033/0036).Measurements. height: c. 23 mm, diameter: c. 21 mm.Remarks. Only a silicone cast of the dorsal part of the shell is available showing prominent spiral sculpture and granulose spiral cords on the spire whorls. Despite the fragmentary preservation, the specimen agrees well with strongly sculptured specimens of Semicassis bisulcata as reviewed by Beu .Measurements. height: 78 mm, diameter: 58 mm.Remarks. A single poorly preserved specimen is available with remnants of sculpture on the spire. Outline and the characteristic very coarse and prominent spiral cords on the low angled sutural ramp agree fully with the Western Tethyan/Proto-Mediterranean Melongena lainei.Stratigraphic and geographic distribution. Melongena lainei is widespread during the Chattian to Burdigalian in the Western Tethys/Proto-Mediterranean and the Paratethys Sea  sykesi  dentata var. sykesi Archiac and Haime—Vredenburg: p. 133.1925 Athleta (Volutospina) sykesi .Measurements. height: 29.2 mm, diameter: 16.1 mm.Description. Small, moderately slender volutid; spire comprising five whorls; early spire conical, forming an angle of c. 80°, later coeloconoid. Sculpture consisting of two rows of small spiny nodes along the shoulder adjoined by two weak spiral threads on sutural ramp. Penultimate and last whorls slightly widening with concave sutural ramp. Upper spiral row of nodes fades out on last whorl, which bears two narrow, smooth spiral cords on concave part of ramp and 24 spiny nodes along shoulder. Below shoulder appear broad axial swellings, crossed by broad spiral cords separated by slightly narrower interspaces; base weakly constricted. Aperture covered by sediment.Remarks. Harzhauser et al. (Athleta (Volutospina) dentata  sykesi from Athleta (Volutospina) dentata. In addition, the higher number of spiny nodes distinguishes A. (V.) sykesi from A. (V.) dentata  and lower Miocene Gaj beds of Sindh in Pakistan and Gujarat in NW-India , pl. 26 Fig. 1 [non Scaphander lignarius  javana Martin: p. 85, pl. 13, Fig. 21.*1879 Scaphander javanus Martin—Vredenburg: p. 11.1925 Scaphander javanus .Measurements. height: 31 mm, diameter: >14 mm.Remarks. A large Scaphander species is abundant in fine-sandstone beds underlying the Irer reef carbonates. The preservation is always poor due to dissolution and only a single cast of a ventral part could be partly extracted from the sandstone. The sculpture of the specimen consists of > 70 densely spaced spiral cords, which tend to bifurcate in the anterior and posterior parts of the shell, separated by narrow, punctate interspaces. This species is frequently reported from the early Miocene Western Indian Province from Gujarat and Kerala in India as Scaphander javanus  or were found as singletons .The first group comprises s Mutvei  are cleaNeritopsis species are exclusively found in reefal environments and submarine caves  sykesi  sykesi  sensu Harzhauser (Mediterranean-Iranian Province (MIP) in the Proto-Mediterranean Sea  anBasterot  in EuropAngaria cf. delphinus .The group of well-known WIP elements from the mudflats and subtidal soft-bottom environments of the northern Indian Ocean is contrasted by a species group, which is unknown from other localities. This species group comprises exclusively species, which were found within the reef carbonates. Such Miocene carbonates, however, are rare along the coasts of eastern Iran, Pakistan and India in the western part of the Indian Ocean (McCall et al. na Fehse , 2015. TGomphotherium-landbridge, respectively, the faunistic influence from the west is negligible. These results coincide with those on corals and foraminifers by McCall et al. (Thus, the early Miocene gastropod assemblages from the northern Indian Ocean are clearly part of the Western Indian Province of the later Indo-West Pacific Region. Despite its close position to the junction to the Proto-Mediterranean Sea or the l et al. .


Trypanosoma (Duttonella) vivax is a major pathogen of livestock in Africa and South America (SA), and genetic studies limited to small sampling suggest greater diversity in East Africa (EA) compared to both West Africa (WA) and SA.Multidimensional scaling and phylogenetic analyses of 112 sequences of the glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) gene and 263 sequences of the internal transcribed spacer of rDNA (ITS rDNA) were performed to compare trypanosomes from tsetse flies from Gorongosa National Park and Niassa National Reserve of Mozambique (MZ), wild ungulates and livestock from EA, and livestock isolates from WA and SA.T. vivax) and TvL (T. vivax-like) evolutionary lineages: 1) Tvv comprises two main groups, TvvA/B  and TvvC/D (exclusively from EA). The network revealed five ITS-genotypes within Tvv: Tvv1 (WA/EA isolates), Tvv2 (SA) and Tvv3–5 (EA). EA genotypes of Tvv ranged from highly related to largely different from WA/SA genotypes. 2) TvL comprises two gGAPDH-groups formed exclusively by EA sequences, TvLA (Tanzania/Kenya) and TvLB-D (MZ). This lineage contains more than 11 ITS-genotypes, seven forming the lineage TvL-Gorongosa that diverged from T. vivax Y486 enough to be identified as another species of the subgenus Duttonella. While gGAPDH sequences were fundamental for classification at the subgenus, major evolutionary lineages and species levels, ITS rDNA sequences permitted identification of known and novel genotypes.Multidimensional scaling (MDS) supported Tvv  contains supplementary material, which is available to authorized users. Trypanosoma (Duttonella) vivax is a major challenge to livestock production in sub-Saharan Africa [T. vivax across Africa and South America (SA) [Animal African trypanosomiasis (AAT) caused by n Africa . This spn Africa –5, as wen Africa , 7. Mechica (SA) , 9.Trypanosoma vivax infections vary in clinical signs and disease severity, and differences in pathogenicity have been linked to breeds of livestock, parasite strains, and geographical locations. In East Africa (EA), there is a wide variation in pathogenicity and lethality. While wild ruminants remain asymptomatic, thus acting as reservoirs, livestock species generally develop significant degrees of anaemia and a range of pathological changes. Severe hemorrhagic syndromes in cattle have been reported in Kenya and Uganda [d Uganda , 11. Furd Uganda .T. vivax causes chronic and debilitating diseases in livestock [T. vivax infections are generally asymptomatic, with beef cattle and water buffalo usually developing chronic disease with very low parasitemia [In West Africa (WA), ivestock , 12. In asitemia , 13. Howasitemia –16.T. vivax [T. vivax was introduced into the New World via cattle imported from WA by European colonisers [T. vivax [T. vivax isolates from cattle clustered either with sequences of isolates from WA or other EA countries regardless of whether they were collected in areas infested by tsetse or not [T. vivax isolates from Zambia  clustered with either WA or divergent EA sequences while all isolates from Ghana clustered with those from WA [Data from earlier studies revealed genetic similarities between SA (Colombian) and WA T. vivax , 18. Thilonisers , 20. In lonisers , 19, 21 lonisers , 23, stulonisers –27. SequT. vivax , 26. In T. vivax . ITS seqe or not . CathepsT. vivax in EA, and probable also in South-Central Africa than in WA/SA [T. vivax diversity. To evaluate genetic repertoires, and any possible links between genotypes and geography, ungulate and vector species, outbreak and endemic areas, and clinical and pathological features, more comprehensive studies with larger sampling are still required. Data on trypanosome diversity in conservation areas are fundamental to wildlife conservation, to track the spread of parasites to bordering farming zones, and to monitor potential pathogenicity in livestock of trypanosome species/genotypes sustained by natural transmission cycles.Characterization of a limited sampling suggests a higher diversity of in WA/SA , 24–26. T. vivax in MZ and to infer phylogenetic relationships among EA, WA and SA T. vivax populations. To achieve this, we determined gGAPDH, and ITS rDNA sequences from a comprehensive sampling of T. vivax isolates from tsetse flies captured at the Gorongosa National Park (GNP) and Niassa National Reserve (NNR), and from wild ungulates and livestock from MZ. The sequences determined in the present study were then compared to those available from other EA countries, and with sequences obtained herein or in previous studies of T. vivax from livestock across WA and SA.The present study aimed to assess the genetic repertoire of 2  located in the centre of MZ in the Province of Sofala, at the southern end of the Great African Rift Valley  (18°45′58″S 34°30′00″E) and Niassa National Reserve (NNR) . GNP comprises an area of ~4000 km2 located in the northern Provinces of Cabo Delgado and Niassa bordering Tanzanian Game Reserves  barcoding [Tsetse flies were captured in highly infested areas of GNP  and NNR (2013 and 2014)  and Maputo (South) of MZ. Livestock was raised having rare contact with wild ruminants, except in areas bordering conservation areas and game reserves, where wild animals and livestock frequently interact  [T. vivax using FFLB were submitted to whole genome amplification (WGA) using the REPLI-g UltraFast Mini Kit .Aliquots (~1.0 ml) of blood were collected using EDTA-treated tubes, preserved in ethanol and employed for DNA preparation as previously described , 20, 21.ATL-PCR) , 30. We T. vivax and T. vivax-like obtained from tsetse flies had a lower sensitivity compared with FFLB and required cloning and sequencing of several clones from each sample. Sequences obtained were deposited in GenBank (Table PCR amplification of gGAPDH sequences from DNA from tsetse flies and blood samples was conducted using a nested PCR method . Each amhttp://www.R-project.org). MDS was performed using the Bios2mds package [To provide a visual representation of the level of similarity across sequences in the dataset, we carried out multidimensional scaling (MDS) analyses plotted in 2D and 3D using the R software platform , Pycnomonas (T. suis) and Nannomonas . The final alignment was analysed using maximum parsimony (P) and the program PAUP*4.0b10, and maximum likelihood (ML) using RAxML with GTRGAMMA (500 maximum parsimony starting trees), model parameter estimated in RAxML over the duration of the tree search, and nodal support estimated with 500 bootstrap replicates.The whole gGAPDH sequences dataset was aligned and identical sequences from the same sample were removed from the final alignment, which also included sequences from GenBank of Sequences of ITS rDNA (ITS1 + 5.8S + ITS2) obtained from DNA of tsetse and blood samples were aligned with sequences available in GenBank  of Glossina pallidipes. Similar prevalence of T. vivax and T. vivax-like was found for the two species of tsetse flies .Barcoded tsetse flies from GNP and NNR were identified as T. vivax and T. vivax-like in 151 fly mouthparts examined by FFLB ranged from 19.2% (29 positive flies of 151 examined) in GNP to 28.6% (93 positive flies of 325 examined) in NNR, and was not significantly different between the two-tsetse species . Altogether, results from GNP and NNR surveys unveiled FFLB profiles that were compatible with T. vivax in the mouthparts of 122 flies (25.6%) out of 476 flies examined, including 12 (2.5%) flies positive for T. vivax and T. vivax-like in both mouthparts and guts, and 8 (1.6%) flies showing only positive guts. Detection of T. vivax in fly guts suggests that parasites can be detected using sensitive diagnostic methods from a fresh blood meal or from contamination during fly dissection. However, as expected, FFLB of most gut samples did not reveal T. vivax or T. vivax-like . Isolates considered positive for T. vivax differed in some peaks of the whole FFLB profiles. However, Duttonella exclusive fluorescent peaks were always present.Prevalence rates of n = 5), reedbuck (n = 2) and warthogs (n = 7) the results were negative.Analysis of MZ tsetse flies using TviCATL-PCR revealed infections in 43 out of 235 tsetse flies (18.2%) tested using this method, demonstrating that the sensitivity of this PCR method is inferior, but rather comparable to FFLB . In addition, TviCATL-PCR detected infection in 10 Cape buffalo  and one wildebeest . In Sunni antelopes , WA (Nigeria) and EA (MZ) [T. vivax Y486), and a broader assemblage of sequences referred to as Tv-like (TvL) lineage, comprising a consistent cluster forward referred as TvL-Gorongosa, formed by groups A-D of isolates diverging from T. vivax Y486 by 4.8 to 5.7% (average of 5.2%) of gGAPDH sequence divergence. In addition, ~8.0% of sequences obtained from GNP and NNR tsetse flies remained unclustered, suggesting that they represent additional TvL lineages  , 26. In ges Fig. .Fig. 2MuTo obtain better resolved MDS clustering for the assessment of inter- and intra-cluster diversity, the dataset from each main cluster was analysed separately Fig. . Tvv unvThe highly divergent TvL sequences were arranged in four main clusters: TvLA  from MZ, Kenya, Tanzania and Ethiopia. The analysis included a large set of ITS sequences from WA (59 sequences from cattle) and SA , most of them determined in the present study  and Tvv2 (SA) genotypes. However, to date, not a single ITS rDNA sequence from Africa was identical to those of Tvv2, which comprises all the 39 samples from Brazil, Venezuela, Colombia and French Guiana examined in our study. Our findings support the hypothesis that Tvv2 may be a bottlenecked genotype that recently diverged from closely related genotypes escaped out of WA. In a study based on microsatellite markers, we also demonstrated that  than SA .T. vivax is cyclically transmitted by tsetse flies and mechanically transmitted by other biting flies [T. vivax is only mechanically transmitted by non-tsetse biting flies. Genomic studies comparing Venezuelan and WA T. vivax Y486 evidenced a drastic process of mitochondrial genome degradation in SA isolates, whereas the African T. vivax Y486 exhibited entirely functional mitochondria necessary for the development in tsetse flies [To date, all TvL isolates came from tsetse-infested areas in EA. Although an early study carried out in Ethiopia suggested that isolates from tsetse-free areas clustered with SA/WA genotypes, while those from tsetse-infested areas clustered with EA genotypes, analysis based on polymorphic ITS sequences did not support this hypothesis , 27. In ng flies , 7, 27. se flies .Duttonella. Molecular data provided herein corroborate differences between WA and EA T. vivax and among EA isolates regarding their morphology, infectivity to tsetse flies, pathogenicity and antigenic relationships. In the last taxonomical revision of Trypanosoma [T. (Duttonella) vivax vivax (= T. vivax) that is the type-species of the subgenus [T. (Duttonella) uniforme, was recognised as a valid species of Duttonella, characterised by small-sized blood forms. This species was reported in cattle, antelope, buffalo and giraffe, widespread in Uganda and Congo, and reported in Zululand, Tanzania and Ethiopia. T. (Duttonella) vivax ellipsiprymni was regarded as a subspecies due to the morphological intergradation of its blood forms with those of T. vivax [Trypanosoma uniforme and T. v. ellipsiprymni were thought to be transmitted by G. morsitans, G. palpalis and G. fuscipes in EA [T. vivax-like reported in this and in previous studies [T. vivax-like from a nyala antelope in MZ showing large blood trypomastigotes morphologically resembling those of T. v. ellipsiprymni [T. vivax Y486.Our study uncovered a complex of species/genotypes within the subgenus panosoma , in addisubgenus , T.  suis, which was initially phylogenetically positioned near T. brucei and provisionally named Trypanosoma sp. Musubugwe, was molecularly validated using DNA recovered from archived blood smears [Duttonella can serve as DNA source for comparison with species candidates uncovered in this and other studies.Recently, d smears . IsolateG. m. morsitans and G. pallidipes. Probably because G. morsitans was highly predominant in our study, a larger repertoire of trypanosomes was found in this species. Trypanosomes from Tanzanian G. pallidipes and G. swynnertoni [T. vivax developed in a range of Glossina species. In contrast, EA isolates of Duttonella likely prefer sympatric tsetse flies. Nevertheless, G. pallidipes was shown to be equally competent in experimentally transmitting T. vivax from Kenya and Nigeria [We identified different genotypes of Tvv and TvL in nnertoni , 26 slig Nigeria , 55. The Nigeria  feeding T. vivax populations in WA, high antigenic cross-reactivity was reported for isolates from The Gambia and Nigeria, but not between these and distantly related Kenyan isolates. In addition, isolates from Kenya differed in resistance to drugs [T. vivax isolates from areas that were or were not infested by tsetse flies in Ethiopia did not include molecular characterization of the isolates [T. vivax infected cattle in Kenya, Ethiopia and Uganda, genetic diversity of a comprehensive sampling remains to be investigated.Corroborating the close relationships between to drugs , antigento drugs , 11. Stuisolates . DespiteT. vivax and T. vivax-like gGAPDH and ITS rDNA sequences uncovered two major evolutionary lineages, constituted by several genotypes, within the subgenus Duttonella: T. vivax (Tvv) composed of five genotypes, and T. vivax-like (TvL) constituted by TvL-Gorongosa, an assemblage of seven genotypes, in addition to genotypes that likely belong to other TvL lineages. Phylogenetic inference supports the rising of TvL-Gorongosa to the species status. We detected TvL-Gorongosa in G. m. morsitans and G. pallidipes, as well as in wild and domestic ungulates, in MZ, Tanzania, Kenya and Ethiopia. However, before proposing any new species attempts to compare this new lineage with previously reported species and subspecies of Duttonella [Duttonella trypanosomes. Genetic diversity was noticeable in EA wildlife conservation areas, where wild ungulates serve as reservoirs for a large diversity of Tvv and TvL genotypes that are transmitted by a range of tsetse species. Not a single TvL isolate was identified in WA, where T. vivax population shown to be quite homogeneous, even though samples examined came from The Gambia to Cameroon, over several years. To date, a single genotype was common in livestock from WA and EA. Further studies on the genetic diversity of T. vivax in wild tsetse and ungulates in WA are required to evaluate whether the lower diversity found in WA was because all isolates examined were obtained from cattle. Data from this study provide additional support for the hypothesised bottlenecked SA population, recently imported from WA. The geographical origin of any pathogenic trypanosome is an issue of high interest, and information on the genetic diversity is fundamental to understand its distribution. We suggested an EA origin for Duttonella species, and hypothesised the spread of Tvv genotypes adapted to livestock, and transmitted either cyclically by tsetse or mechanically by other flies, following historical livestock dispersion routes. Any data on the genetic and spatial structure is valuable for assessing possible links between genotypes and vertebrate hosts, vector species, pathogenicity, and drug resistance of T. vivax and T. vivax-like.Our comparative study of a large sample of ttonella  are highAdditional file 1: Table S1.Trypanosoma vivax isolates from Africa, including the host species, geographical origin and groups/genotypes defined by gGAPDH and ITS rDNA analyses. TvL-G: TvL-Gorongosa. (DOCX 29 kb)Additional file 2: Table S2.Trypanosoma vivax isolates from South America, including the host species, geographical origin and groups/genotypes defined by gGAPDH and ITS rDNA analyses. (DOCX 19 kb)Additional file 3: Figure S1.Trypanosoma vivax and T. vivax-like isolates. South American and West African isolates shared highly conserved sequences, exhibiting only punctual polymorphisms. The polymorphic East African isolates revealed blocks of nucleotides that were unique for each genotype, as well as conserved segments shared by closely related genotypes. The reference isolate/sequence representing each genotype is underlined. ITS sequences were deposited in GenBank (accession numbers in Additional files The alignment of ITS1 and ITS2 rDNA sequences from


Bombyx mori).Long non-coding RNAs (lncRNAs) may play critical roles in a wide range of developmental processes of higher organisms. Recently, lncRNAs have been widely identified across eukaryotes and many databases of lncRNAs have been developed for human, mouse, fruit fly, etc. However, there is rare information about them in the only completely domesticated insect, silkworm  from previous studies. Then, we organized them into a comprehensive and web-based database, BmncRNAdb. This database offers a user-friendly interface for data browse and online analysis as well as the three online tools for users to predict the target genes of lncRNA or miRNA.http://gene.cqu.edu.cn/BmncRNAdb/index.php.We have systematically identified and collected the silkworm lncRNAs and constructed a comprehensive database of the silkworm lncRNAs and miRNAs. This work gives a glimpse into lncRNAs of the silkworm and lays foundations for the ncRNAs study of the silkworm and other insects in the future. The BmncRNAdb is freely available at The online version of this article (doi:10.1186/s12859-016-1251-y) contains supplementary material, which is available to authorized users. The ENCODE project estimates that 62–75 % of the human genome are transcribed, but only 2 % of the transcripts can be translated to proteins , 2. The Xist (X-inactive specific transcript) is a long non-coding RNA on the X-chromosome and takes part in inactivation of X-chromosome during the early developmental process of female embryo [Acal acts as a novel negative dorsal closure regulator during Drosophila embryogenesis and Lnccov1 is involved in the autophagic cell death of ovarioles in Apis mellifera [Long non-coding RNAs (lncRNAs) are transcripts of DNA that are usually considered to be > = 200 nt (nucleotide) and do not have apparent coding capacity –10. LncRe embryo , 19. In e embryo , 20–25. ellifera , 27. TheBombyx mori, is one of important model organisms for Lepidoptera, more and more transcrptomic resources are available for the silkworm. The ncRNAs, especially microRNAs (miRNAs) were identified in the silkworm by Solexa sequencing [Fben-1  was identified in female-brain and may be involved in sexually dimorphic brain functions [The domesticated silkworm, quencing . In addiquencing . As one unctions . Althougunctions , 31.http://gene.cqu.edu.cn/BmncRNAdb/index.php.Moreover, many databases on the information of lncRNA have been developed such as NONCODE, lncRNAdb, LncRBase, DeepBase –35, but The BmncRNAdb database implementation is based on the Gentoo Linux system with the tools of Apache 2.0 , PHP 5.4http://metazoa.ensembl.org/) [http://www.ncbi.nlm.nih.gov/sra) [http://www.uniprot.org/) [ftp://ftp.ebi.ac.uk/) [New version of the silkworm genome sequence was downloaded from the silkworm genome database, SilkDB v2.0 . The silbl.org/) . All thegov/sra) –65. The gov/sra) . Non-redgov/sra) . A comprot.org/) . The cur.ac.uk/) .Two types of data from the silkworm were used for identification of the silkworm lncRNAs. The first is the silkworm RNA-seq data. Forty-one RNA-seq datasets were published by other research groups before January 15(th), 2015 and four RNA-seq datasets were produced by our laboratory  Toolkit of NGS (Next-Generation Sequencing) is used to control the reads quality of forty-five RNA-seq datasets . High-quFben-1, is identified by our pipeline. This shows the reliability of our pipeline.LncRNAs are usually considered to have length > =200 bp and ORFs (open reading frame) < = 100 aa (amino acids) , 43, 70.Apis mellifera (Hymenoptera) lncRNAs, the highest sequence conservation  and Apis cerana (Hymenoptera) lncRNAs. However, the silkworm lncRNAs have low sequence conservation with the Drosophila melanogaster, Anopheles gambiae and Nilaparvata lugens lncRNAs. Furthermore, the expressions of the genes within 2 kbp neighbor regions (2 kbp upstream and 2 kbp downstream) of the putative silkworm lncRNAs are not significantly correlated with the expressions of lncRNAs (Spearman test)  Fig.  and g.FiThe silkworm microRNAs were comprehensively identified in the whole body, anterior or middle and posterior silk glands by next generation sequencing technology , 29. Ther = 0.017, P-value = 0.62) . The database contains six functional sections, data browse, keywords search, Blast alignment, lncRNA target gene discovery, miRNA target gene discovery and data download.Using the pipeline in Fig. 62) Fig. . This isIn the left navigation, clicking the ‘Browse’, users can browse the information of lncRNAs including lncRNA name, scaffold, start position, end position, exon number and length Fig. . By clicThe online analysis tools about lncRNAs and miRNAs are provided in the BmncRNAdb to facilitate functional research of lncRNAs and miRNAs. Four user-friendly online analysis tools are available for users including Blast + , LncTar BmncRNAdb offers the download section for users to obtain all the silkworm lncRNA sequences, miRNA sequences and example data. In the help section, a guide manual is shown to help the users to learn how to better use the BmncRNAdb for their own research. In addition, under the left navigation, several useful or famous database resources about ncRNAs are collected in the BmncRNAdb related links. Our group will continue to collect more information on the silkworm ncRNAs and add more useful online tools about the functional research of ncRNAs to the BmncRNAdb in the future.Bombyx mori). Through the BmncRNAdb database, users can browse and search for the detail information of lncRNAs and miRNAs in the silkworm. In addition, this database provides three online tools for users to find the target genes of an lncRNA and miRNA. BmncRNAdb will facilitate the ncRNA research of the silkworm and other insects in the future. Moreover, the availability of the complete set of lncRNAs from the silkworm will improve the comparative and evolutionary analyses of lncRNAs among different Lepidoptera or other insect species.We have systematically identified and collected 6281 silkworm lncRNAs using the RNA-seq data and unigenes. We also collected 1986 silkworm miRNAs that were predicted by NGS. Integrating these lncRNAs and miRNAs data, we have constructed a comprehensive lncRNAs and miRNAs database (BmncRNAdb) for the silkworm (Database: BmncRNAdbDatabase homepage:http://gene.cqu.edu.cn/BmncRNAdb/index.phpOperating system(s): LinuxProgramming language: PHP, CGI, JavaScript, PerlOther requirements: MySQL, ApacheThe database is freely available without restrictions for use by academics and non-commercial researches. Inquiries concerning the database may be directed to zezhang@cqu.edu.cn or huaxia2033@126.com.


Southeast Asia harbors a unique and diverse freshwater fauna of Mesozoic origin, which is under severe threat of extinction because of rapid economic development and urbanization. The largest freshwater basins of the region are certainly the primary evolutionary hotspots and they attract the most attention as key biodiversity areas for conservation. In contrast, medium-sized rivers are considered low-importance areas with secondary biodiversity, whose faunas originated via founder events from larger basins during the Pleistocene, although such a scenario has never been tested by using a phylogenetic approach. In this investigation, we used freshwater mussels (Unionidae) as a model to estimate the levels of endemism within the Sittaung, a little-known remote basin in Myanmar, compared with the surrounding larger rivers . We discovered that the Sittaung represents an exceptional evolutionary hotspot with numerous endemic taxa of freshwater mussels. On the basis of our extensive dataset, we describe two new tribes, two genera, seven species and a subspecies of Unionidae. Our results highlight that medium-sized basins may represent separate evolutionary hotspots that harbor a number of endemic lineages. These basins should therefore be a focus of special conservation efforts alongside the largest Southeast Asian rivers. Climate change may increase the effects of human impacts, especially for taxa with low abundance and restricted ranges, and may trigger multiple local extinctions8. Our understanding of spatial biodiversity patterns across freshwater basins is very limited because the systematics of many groups are not developed, including the important invertebrates such as bivalves and gastropods13. In Southeast Asia, the lack of reliable taxonomic information precludes producing the national maps of freshwater biodiversity hotspots, which is a task of great importance for conservation planning9.In the modern period of the sixth mass extinction (Anthropocene), freshwater biodiversity is under severe threat because of increasing anthropogenic pressure, which leads to habitat degradation, water pollution, keystone species declines, and the homogenization of faunas17. The Unionidae most likely originated in Southeast and East Asia in the Jurassic, with subsequent expansions into other landmasses9. In several major Asian river systems , exceptional intra-basin radiations of the Unionidae were discovered, which suggests that these basins may be considered ancient (long-lived) rivers that have existed throughout the Cenozoic18. However, the freshwater mussel faunas of Asia have attracted little attention from scientists compared with those from Europe and North America17. Although the importance of freshwater mussels in tropical ecosystems is still poorly known, they could play an essential role as biofilters in polluted water bodies19. Several species are successful invaders, and have spread beyond their native ranges together with the introduction of their host fishes and may threaten native communities21. Finally, freshwater mussels are important objects for the ornamental pet trade, pearl cultivation and food markets across Asian countries24.Here, we use mussels in the family Unionidae, or naiads, as a model group for the assessment of spatial patterns of freshwater biodiversity across western Indo-China. This is the most species-rich bivalve family, with ~620–680 extant species25. In western Indo-China, most historical samples were collected from the Irrawaddy, Salween, Pegu, Tavoy and Great Tenasserim river catchments48. In contrast, nominal taxa from the Sittaung, a medium-sized river basin in Myanmar, were not described. In the 1880s, Leonardo Fea, an Italian naturalist and traveler, collected mussels from several tributaries of the Sittaung49. Fea’s collection was deposited in the Museo Civico di Storia Naturale di Genova . Since then, none of the freshwater mussels have been collected from the basin. Moreover, for some enigmatic reason Fea’s samples from the Sittaung were not used for the description of any unionid taxon50.The taxonomy of freshwater mussels of Southeast Asia is complicated, because many nominal taxa were described from this region on the basis of conchological features, including small differences in shell shapeet al.9 showed that each large river system of this region is a separate evolutionary hotspot harboring a unique endemic naiad fauna. In the present investigation, we expand this sample to estimate the levels of endemism within the Sittaung compared with the surrounding larger rivers . From the phylogenetic and morphological studies of the mussel samples, we describe seven new species and a single subspecies, which are ancient endemic lineages of the Sittaung. Additionally, we found two new tribes and two new genera, which are described herein. Our findings highlight that the medium-sized basins may represent separate evolutionary hotspots that should be considered in future conservation planning for freshwater biodiversity in Southeast Asia.From the comprehensive phylogenetic study of the Unionidae across the primary basins of the Oriental Region, Bolotov Indonaia and Radiatula) into a single MOTU  contains 469 specimens of Unionidae, including 403 specimens from the Oriental Region Type Genus: Comments: This subfamily includes at least five valid tribes: Parreysiini Henderson, 1935, Coelaturini Modell, 1942, Lamellidentini Modell, 1942, Oxynaiini Starobogatov, 197017 and Leoparreysiini Vikhrev, Bolotov et Kondakov, a new tribe described here.Distribution: Africa, South and Southeast Asia54.Tribe Leoparreysiini Vikhrev, Bolotov et Kondakov tribe nov.Leoparreysia Vikhrev, Bolotov et Aksenova gen. nov.Type Genus: Comments: This tribe includes only the genus Leoparreysia Vikhrev, Bolotov et Aksenova gen. nov.Diagnosis: Shell very thick, shape of round, umbo rather prominent and situated near the anterior end. Pseudocardinal teeth well developed; laterals convex and thick. Anterior adductor scar round and very deep.Distribution: Western Indo-China.Leoparreysia Vikhrev, Bolotov et Aksenova gen. nov.Genus Leoparreysia canefrii Vikhrev, Bolotov et Kondakov sp. nov.Type Species: Comments: We assigned eleven species to the genus, including a new species from the Sittaung River basin (Table in Table .Etymology: This genus is named after Leonardo Fea (1852–1903), an adventurous Italian naturalist, who sampled freshwater mussels from unexplored areas of British Burma (Myanmar).Diagnosis: The same as for the tribe.Distribution: Western Indo-China.Leoparreysia canefrii Vikhrev, Bolotov et Kondakov sp. nov.Figures Type material: Holotype RMBH biv254_4: Myanmar: Sittaung near Taungoo, 26.xi.2016, Vikhrev leg. Paratypes: the type locality, 6 specimens , 25–26.xi.2016, Vikhrev leg.Etymology: This species is named after Cesare Maria Tapparone-Canefri (1838–1891), an Italian malacologist, who described numerous mussel taxa from British Burma (Myanmar).Diagnosis: The new species is similar to L. olivaceacomb. nov. and L. tavoyensiscomb. nov. but differs in a more inequilateral shell shape, specific shell sculpture with zigzag ridges, deeper umbo cavity, and fixed nucleotide substitutions  20.0–76.0 mm, height (SH) 14.6–56.4 mm, width (SW) 9.2–33.6 mm. Shell sculpture strong. Periostracum dark-olive to yellowish-brown; nacre white, sometimes with yellow spots. Umbo pronounced; beak sculpture strong. Left valve with two short curved lateral teeth and two striate pseudocardinals. Right valve with a single curved lateral tooth and two distinct pseudocardinals; anterior tooth strongly indented, massive, posterior tooth small and lamellar. Umbo cavity deep. Anterior adductor scar well-marked, very deep, funneled; posterior scar shallow.Distribution: Sittaung River.Tribe Oxynaiini Starobogatov, 1970Oxynaia Haas, 1913 (designated in this study)Type Genus: Comments: This tribe includes at least three phylogenetically distant genus-level clades: Radiatula Simpson, 1900 (see below), Indonaia Prashad, 1918 stat. res.  and Oxynaia Haas, 1913 . The latter clade may belong to another, possibly undescribed genus, but the Oxynaia jourdyi sequences are not available. A single Indonaia species and three Oxynaia taxa were found in western Indo-China Type Species: Comments: Five species of the genus are recorded in western Indo-China, including a new species from Sittaung , 25–26.xi.2016, Vikhrev leg.Etymology: This species is named after Henri Mouhot (1826–1861), a French naturalist and explorer, who travelled across Siam, Cambodia and Laos.Diagnosis: The new species is similar to R. aff. bonneaudii sp.1, but differs by a less deep and rounded anterior adductor scar, a well-marked posterior scar, and fixed nucleotide substitutions Type Genus: Comments: This tribe includes at least two distant genus-level clades: Lamellidens Simpson, 1900 and Trapezidens Bolotov, Vikhrev et Konopleva gen. nov.Distribution: South and Southeast Asia45.Lamellidens Simpson, 1900Genus Unio marginalis Lamarck, 1819 Type Species: Comments: A large genus, which includes numerous nominal taxa52 and multiple cryptic DNA lineages, particularly from India9. In contrast, the species diversity of Lamellidens in western Indo-China was largely overestimated, as we were able to find only four species there , Myanmar: Sittaung, reservoir at Yetho River, 3 specimens , Myanmar: Sittaung, fishing ponds near Taungoo, 1 specimen (RMBH biv247_10), Myanmar: Sittaung, Myit Kyi Pauk Stream, 1 specimen (RMBH no. biv250_13), 23–26.xi.2016, Vikhrev leg.Etymology: This species is named after Dr. Rolf Arthur Max Brandt (1917–1989), a famous German malacologist, who studied the mussels of Southeast Asia.Diagnosis: The new species is similar to L. savadiensis, but differs by a smaller shell, shallow anterior and posterior adductor scars, and fixed nucleotide substitutions Type Species: Comments: A small remarkable genus with three species: T. exolescenscomb. nov., T. obesastat. res. et comb. nov. (with three subspecies) and T. scutumcomb. nov. , Myanmar: Sittaung near Taungoo, 1 specimen (RMBH biv255_1), 26.xi.2016, Vikhrev leg.Etymology: This subspecies is named after Leonardo Fea (1852–1903), an Italian naturalist.Diagnosis: The new subspecies differs by an almost straight dorsal margin without a wing, weakly developed adductor scars, a more oblong shell shape, and fixed nucleotide substitutions Type Genus: Comments: This remarkable subfamily, which was restored by Bolotov et al.,9 includes the two valid tribes: Pseudodontini Frierson, 1927 and Pilsbryoconchini Bolotov, Vikhrev et Tumpeesuwan tribe nov. Frierson55 (p. 67) did not provide any description of the subfamily, but noted that “The above species [Gonidea angulata ] is usually without ‘teeth’, but sometimes bears large high teeth, one in each valve, strikingly alike to the Pseudodon cambodgensis [sic.], and the outward facies of the two species are quite similar, so that it might be that the species in fact a member of the Pseudodontinae.”Distribution: Southeast and East Asia45.Tribe Pseudodontini Frierson, 1927Pseudodon Gould, 1844 Type Genus: Comments: This tribe includes the single genus Pseudodon Gould, 1844. The genera Trigonodon Conrad, 1865  and Indopseudodon Prashad, 1922  are considered synonyms of Pseudodon on the basis of conchological features.Distribution: Western Indo-China.Pseudodon Gould, 1844Genus Anodon inoscularis Gould, 1844 Type Species: Comments: This genus contains seven species, including two new species from Sittaung , 23.xi.2016, Vikhrev leg.Etymology: This new species is dedicated to Dr. Arthur Bogan, a famous American malacologist, who developed the basic principles for the conservation of freshwater mussels worldwide.Diagnosis: The new species is similar to P. avae and P. manuelisp. nov., but differs by a straighter ventral margin, massive hinge plate, and fixed nucleotide substitutions , 25.xi.2016, Vikhrev leg.Etymology: This species is dedicated to Dr. Manuel Lopes-Lima, a malacologist from Portugal, who developed a recent integrative system for the Unionidae.Diagnosis: The new species is similar to P. boganisp. nov. and P. avae, but differs by less pronounced pseudocardinals, adductor scars not produced, and fixed nucleotide substitutions , Monodontina Conrad, 1853 stat. res. , and Bineurus Simpson, 1900 stat. res. . However, there are three distant, relatively well-supported clades, which may represent distinct genera  and Contradentini Modell, 1942 (see below).Distribution: Southeast Asia, including the Greater Sunda Islands45.Tribe Contradentini Modell, 1942Contradens Haas, 1911 Type Genus: Comments: This tribe includes at least three genus-level phylogenetic clades: Contradens Haas, 1911, Trapezoideus Simpson 1900, and Physunio Simpson, 1900 Type Species: Comments: The genus was established on the basis of conchological features40. We consider this genus as a separate Contradentini clade, which includes taxa inhabiting the rivers of western Indo-China, although the molecular sequences of the type species are not available25. The genus contains at least six species, including two new taxa from the Sittaung   is still uncertain. The two latter taxa may belong to Lamellidens46, but their DNA sequences are not available.ng Table . Among tDiagnosis: Shell rather thin, from a trapezoidal to elongate-elliptic form, the anterior margin narrower than the posterior margin. One lateral tooth and two pseudocardinals in the right valve, two laterals and elongate lamellar cardinal tooth in the left valve. Pseudocardinals reduced or lacking. Anterior adductor scar more or less produced, posterior scar usually less marked. Umbo more or less projected. Older specimens with more elongate shape and flattened umbo.Distribution: Western Indo-China.Trapezoideus nesemanni Konopleva, Vikhrev et Bolotov sp. nov.Figure Type material: Holotype RMBH biv255_2: Myanmar: Sittaung, Tauk Ue Kupt River, 26.xi.2016, Vikhrev leg. Paratypes: the type locality, 4 specimens , 20.iv.2015 and 26.xi.2016, Bolotov, Vikhrev & locals leg.Etymology: This new species is dedicated to Dr. Hasko Friedrich Nesemann, an Austrian malacologist, who made great contributions to the knowledge of molluscs from the Ganga River system.Diagnosis: The new species is similar to T. subclathratus and T. panhaisp. nov., but differs in its very thin shell, shallow adductor scars, and fixed nucleotide substitutions , 17–18.iv.2015, Bolotov & locals leg.Etymology: This new species is dedicated to Prof. Dr. Somsak Panha, a famous Thai zoologist, who described numerous molluscan taxa from Southeast Asia.Diagnosis: The new species is similar to T. subclathratus and T. nesemannisp. nov., but differs by a thicker shell, more developed hinge and umbones, and fixed nucleotide substitutions , L. jenkinsianus , L. lamellatus , L. marginalis , Parreysia corrugata , P. favidens , P. smaragdites , Radiatula bonneaudii , R. caerulea , and R. pachysoma  in the Irrawaddy44 are erroneous. However, the occurrences mentioned above may correspond to morphologically similar but phylogenetically distinct, endemic taxa such as Lamellidens savadiensis, Trapezidens obesa, Leoparreysia spp., Radiatula aff. bonneaudii sp.1, and Indonaia andersoniana. The published records of Lamellidens consobrinus , L. generosus and Physunio ferrugineus from the Irrawaddy10 most likely refer to Lamellidens savadiensis.There is a historical tradition to attribute numerous unionid species from western Indo-China to those described from Indian rivers, which began with the pioneering workset al.9, although the present study suggests that the two largest paleo-Mekong radiations may have had a pre-Cenozoic origin (mean age = 65–71 Ma). Based on our fossil-calibrated phylogeny, the fauna of the Sittaung is related to that of the Irrawaddy but represents a separate evolutionary entity harboring a number of endemic lineages. Our model suggests that the majority of the lineages were separated during the Miocene (mean age = 8.0–22.1 Ma), but the split between Pseudodon avae and the two sister taxa from the Sittaung appears to be more ancient (mean age = 35.3 Ma) , the online registration system for the ICZN. The LSID for this publication is: urn:lsid:zoobank.org:pub:1CC25D33–7DF8–41D5–9817–5285C01A7779. The electronic edition of this paper was published in a journal with an ISSN, and has been archived and is available from PubMed Central.The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature (ICZN), and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank , the British Museum of Natural History, London, UK (NHMUK), and the Museo Civico di Storia Naturale di Genova, Genoa, Italy (MSNG). Additionally, we accessed the images of the types of several nominal taxa at the MUSSELp Database17. The majority of these sequences were sampled from our work on the biogeography of the Oriental freshwater mussels9. New sequences were obtained from 74 specimens belonging to 15 species that were collected from the Sittaung and Irrawaddy river basins, and from a river of the Malay Peninsula , following the manufacturer’s protocol. For molecular analyses we obtained partial sequences of two mtDNA markers, i.e., the cytochrome c oxidase subunit I gene (COI) and the 16S ribosomal RNA (16S rRNA), and a fragment of the nuclear 28S ribosomal DNA (28S rDNA). Primer sequences for PCR are shown in Supplementary Table ® BigDye™ Terminator v. 3.1 reagents kit and run on an ABI PRISM® 3730 DNA analyzer . The resulting sequences were checked by eye using a sequence alignment editor (BioEdit v. 7.2.5)60. A total of 25 mussel species were used as an out-group, including representatives of Margaritiferidae (10 species), Iridinidae (2 species), Etheriidae (1 species), Mycetopodidae (1 species), Hyriidae (9 species) and Trigoniidae (2 species) , East Asia, Europe, Africa and North America . A partition homogeneity test was calculated in PAUP* v. 4.0a151 to confirm the congruence of phylogenetic signals among sequence data sets65. This test revealed that the signals among the data sets are consistent  analyses were performed in MrBayes v. 3.2.669 at the San Diego Supercomputer Center through the CIPRES Science Gateway68. The data set tested was similar under the ML model. The best models of sequence evolution for each partition based on the corrected Akaike Information Criterion (AICc) of MEGA661 are presented in Supplementary Table 70. The effective sample size (ESS) value for each parameter sampled from the MCMC analysis was always recorded as >1300.In phylogenetic analyses, we tested only the dataset with five partitions (3 codons of COI + 16S rRNA + 28S rDNA), because Bolotov 61, which revealed that the null hypothesis of equal evolutionary rate throughout the tree was rejected at a 5% significance level (p < 0.001). Thus, the time-calibrated haplotype-level Bayesian phylogeny was reconstructed in BEAST v. 1.8.4 based on multiple fossil calibration points using a lognormal relaxed clock algorithm with the Yule speciation process as the tree prior73. Calculations were performed at the San Diego Supercomputer Center through the CIPRES Science Gateway68. A fossil-calibrated ultrametric tree was obtained using BEAST v. 1.8.4. We specified similar settings to five partitions (3 codons of COI + 16S rRNA + 28S rDNA) as in the MrBayes analyses, but by using simplified evolutionary models. The HKY model was applied to each partition instead the GTR model, because the prior and posterior ESS values under the GTR model were recorded always <100. This indicates that the GTR model is likely overly complex for our data9. The eight published fossil calibrations were used for timing of the phylogeny9. We designated priors for out-group taxa using a “Monophyly” option of BEAUti v. 1.8.473 as follows: ). Four replicate BEAST searches were conducted, each with 50 million generations. The trees were sampled every 5,000th generation. The log files were checked visually with Tracer v. 1.6 for an assessment of the convergence of the MCMC chains and the effective sample size of parameters70. The first 10–52% of trees were discarded as an appropriate burn-in. All the ESS values were recorded as >250, with exception of two parameters with the ESS >120; the posterior distributions were similar to the prior distributions. The resulting tree files from four independent analyses were compiled with LogCombiner v. 1.8.473. The maximum clade credibility tree was obtained from 31,404 primary trees using TreeAnnotator v. 1.8.473.We estimated the acceptance of a global molecular clock to our multi-gene data set using the maximum likelihood test of MEGA678. MOTUs were separated based on the bPTP model78 to infer putative species boundaries on a phylogenetic input tree inferred from a maximum likelihood (ML) analysis. The ML analysis was conducted based on an alignment of the COI haplotype sequences of Parreysiinae and Pseudodontinae + Rectidentinae groups separately using RAxML v. 8.2.6 HPC Black Box67 at the San Diego Supercomputer Center through the CIPRES Science Gateway68. A unique GTR model was applied for each partition (three codons of COI) with corrections for gamma distribution. Margaritifera laosensis and M. dahurica were used as an out-group for the each group. We used an implementation of the bPTP model thorough online bPTP server (http://species.h-its.org/ptp) with 500,000 Markov Chain Monte Carlo (MCMC) generations and 10% burn-in78. All out-group taxa were removed from the input tree using an appropriate option of the server. The output parameters of the bPTP model for each clade under the highest Bayesian solution were as follows: (i) Parreysiinae: an estimated number of species = 40–88, mean value = 61.1, acceptance rate = 0.46; and (ii) Pseudodontinae + Rectidentinae: an estimated number of species = 45–69, mean value = 53.5, acceptance rate = 0.31. Additionally, MOTUs were obtained using the sPTP (p < 0.001) and mPTP models of Kapli et al.79 thorough online mPTP server (http://mptp.h-its.org). A phylogenetic input tree was obtained from ML analysis, which was conducted based on an alignment of the COI haplotype sequences of the Oriental Unionidae with the two haplotypes of Margaritifera laosensis and M. dahurica as an out-group using RAxML v. 8.2.6 HPC Black Box67.The preliminary delimitation of biological species was based on a molecular approach using the MOTU conceptet al.80. Firstly, we estimate the morphological differences between a new species and closely related (congeneric) taxa. The comparative analysis of the shell morphology was carried out with attention to the structure of the pseudo-cardinal and lateral teeth, muscle attachment scars, shell shape and umbo position25. Secondly, the molecular diagnosis of each new species was provided using the fixed nucleotide differences82, which were estimated for each gene separately using a Toggle conserved sites tool of MEGA6 at 50% level61. For each species, an alignment of congeneric haplotype sequences was performed using the ClustalW algorithm implemented in MEGA661. All the deleterious mutations were retained for the analyses. Additionally, a COI mean p-distance to the nearest neighbor of each species was calculated in MEGA661.In order to diagnose each new species, we followed a two-step procedure of Delić The sequences generated under this study are available from GenBank. Accession numbers for each specimen are presented in Supplementary Table Supplementary Info