AbstractDoryphora species reveal that all are oviparous and feed on vines in the Apocyanaceae. Mothers in the two subsocial species defend eggs and larvae by straddling, blocking access at the petiole and greeting potential predators with leaf-shaking and jerky advances. A less aggressive form of maternal care is found in two Platyphora and four Proseicela species associated with Solanaceae, shrubs and small trees. For these and other morphologically similar taxa associated with Solanaceae, genetic distances support morphology-based taxonomy at the species level, reveal one new species, but raise questions regarding boundaries separating genera. We urge continued study of these magnificent insects, their enemies and their defenses, both behavioral and chemical, especially in forests along the eastern versant of the Central and South American cordillera.A summary of literature, documented observations and field studies finds evidence that mothers actively defend offspring in at least eight species and three genera of Neotropical Chrysomelinae associated with two host plant families. Reports on three  Arthropod parents influence the survival prospects of offspring in a multitude of ways. When parents bring resources to larvae, guide larvae to resources or actively shield offspring from predators and parasitoids, they are engaging in subsocial behavior , 1975. TColeoptera include numerous examples of independently evolved subsocial behavior where the importance of competition, resource provisioning and defensive parental behavior can be examined (Chrysomelinae) and the tortoise beetles (Cassidinae), groups possibly more vulnerable to predators and parasitoids due to slow-moving and exposed immature stages  and 20 µl proteinase K with a sterile pestle, vortexed for 10 s and incubated overnight at 55° C. Following incubation, 200 µl AL lysis buffer (Qiagen Inc.) was added and the sample was heated at 70° C for 10 min, then 200 µl molecular grade ethanol was added to each sample. This mixture was then pipetted into a DNeasy mini spin column and centrifuged at 8000 rpm (~6000 g) for 1 min, then the flow-through and collection tube were discarded. The DNeasy mini spin column was placed in a new 2-ml collection tube and 500 µl wash buffer AW1  was added, the sample was centrifuged for 1 min at 8000 rpm, then the flow-through and collection tube were discarded. Again a new collection tube was used, 500 µl wash buffer AW2  was added and the sample centrifuged for 3 min at 14,000 rpm ; the collection tube was then discarded. The mini column was placed in a 1.5-ml tube and 200 µl AE elution buffer  was added, the sample was incubated for 2.5 min at room temperature, and the sample centrifuged for 1 min at 8000 rpm (~6000 g). Extractions were held at -20° C between use, and at -80° C for long-term storage. The primers: C1-J-1718F  and C1-N-2191  (2 (25 µM), 1µl nucleotide mix (8 mM each), 0.8 µl dimethyl sulfoxide 5%, 1 µl each primer (20 mM), 0.2 U Taq DNA polymerase  plus sterile water. The PCR cycling conditions were: 94° C for 2 min, 10 cycles of 94° C for 30 s, 46° C for 30 min, 72° C for 45 min, then 24 cycles of 94° C for 30 s, 48° C for 30 min, 72° C for 45 min, and finally 72° C for 10 min and 10° C for 2 min.Adult T TC-3’)  were useForward and Reverse sequences were combined and reconciled in Sequencher v5  and trimmed, leaving a single 472 bp fragment, which was then translated to amino acids and found free of stop codons. Sequences from ten species were combined with three sequences from GenBank creating an ingroup of 12 species  and a single outgroup species. Where possible we included two separate individuals from the same population. New sequences were deposited in GenBank under accession numbers in PageBreakPageBreakPageBreakhttp://species-id.net/wiki/Doryphora_paykulliDoryphora paykulli adult following a tightly arranged group of larvae moving between leaves on their food plant near Chiriquí Grande, Bocas del Toro Province  documents the presence of the species in the Caribbean as well as in the Pacific lowlands of Panama and provides the first unequivocal record of subsocial habits for the species.According to Doryphora paykulli adults were observed  during late April and early May of 2005 moving slowly and feeding among low vegetation. Pairs of individuals were observed interacting aggressively on the small leaf fragments remaining on the host plant, Prestonia seemannii Miers (Apocynaceae) under late dry season conditions along trails in the Parque Metropolitano , Panama City. Whether these were contests over resources or precopulatory courtship is unclear, however, analysis of video taken of one of these interactions shows the use of the mesosternal horn in dislodging a competitor, much as described by Doryphora sp. near punctatissima. Adult females were discovered during mid-May of both 2005 and 2007 in close proximity to eggs attached to the underside of cupped, newly expanded Prestonia seemannii leaves . Eggs measured 3.5 × 0.9 mm and at first were an opaque, cherry-red, with the chorion becoming transparent and larvae distinguishable as development progressed. Clutches initially contained ten eggs with roughly ten new eggs added each day over the course of 3 to 5 days. The female became noticeably more defensive the second day of oviposition, straddling the eggs and jerking from side to side apparently in response to movement by the observer.Subsequently, individual i leaves  on the wPageBreakonce-developing larvae visible under close inspection (PageBreakmothers might provide a non-invasive means of assessing defensiveness. On one occasion an Ectatomma tuberculatum (Olivier) worker was observed to pass by the base of the petiole, eliciting aggressive shaking of the natal leaf and short charges, after which the ant reversed course and departed that portion of the plant. As larvae became larger and began moving between leaves the intensity of the mothers’ reactions to foreign stimuli appeared to subside. Increasingly, mothers were seen feeding on leaves and leaf petioles, rather than guarding, as larval development proceeded.Larval emergence began on days 6 and 7 following first oviposition with clutches (n=3) at that time containing from 40–50 eggs. Within 24 hours after larvae began to emerge many of the original eggs were missing, apparently consumed by early-emerging larvae such that clutches were reduced to 8, 15 and 20 surviving larvae accompanied by some intact, opaque, less-developed eggs and opened eggs with red-colored residues of spection . One dayspection . While gDoryphora sp. near punctatissima by Doryphora paykulli in Panama and Doryphora sp. near punctatissima in Colombia  reticulata (Fabr.) in the cerrado of south-central Brazil  in 2005 clearly show behaviors strikingly similar to that observed in Doryphora paykulli in Panama. Females oviposit on the underside of partially-expanded, apical leaves of Prestonia tomentosa (Apocynaceae)  . Larvae ynaceae) , but unlthe soil . Indeed,l Brazil , Dorypho conviva  are incoDaccordi, 1994http://species-id.net/wiki/Platyphora_selvaChrysomelinae, reports of subsociality until recently were limited to a single species studied at the La Selva Field station in the Atlantic lowlands of Costa Rica  heteroclita Sendtm. (Solanaceae) in the Atlantic lowlands of Costa Rica. His observations were remarkable in first describing how females of this species tightly guarded offspring by straddling. By removing mothers from roughly half of the families, he was able to demonstrate that guarding was highly effective in preventing predation by the gigantic ponerine ant, Paraponera clavata Fab. The importance of maternal defenses in reducing losses to parasitoids, however, was not investigated. Further, it was noted that mothers always guarded groups of four or fewer larvae; but eggs of the beetle were never observed during the study. From observations of related taxa (see below) we now suspect that Platyphora selva is not oviparous, but instead deposits temporally isolated clutches of four larvae. This inference remains to be documented and is based on the habits of the morphologically similar species, Platyphora microspina, which occurs widely (but rarely) in neighboring Panama. Regrettably, sequence data are not yet available for Platyphora selva.http://species-id.net/wiki/Platyphora_microspinaPageBreakPlatyphora microspina was initially observed on Cerro Campana , along a ridgeline approximately 50 m west and up-slope from the Podocarpus trail in July 1999 . Large numbers of adult and immature beetles were observed feeding on Markea megalandra , a woody hemiepiphyte which grows within the canopy of forests at elevations of 1000–2000 m in Western and Central Panama http://species-id.net/wiki/Proseicela_vittataPageBreakPageBreakProseicela vittata , 11–18 April 2010, 25–31 January 2011 and 19 June–2 July 2012. All individuals and family groups were found on a single species of host plant, Solanum morii S. Knapp, a 1-3 m shrub with glabrous leaves and pendulous green and white fruit and a colonist of disturbed areas  the period of overlap is brief. Normally, individual larvae within cohorts were remarkably similar in size , but eventually larvae pushed by and began traversing nearby stem and petioles solitarily or in small groups. Commonly families split into two or more separated feeding groups at this stage, with the mother usually remaining with a larger group. Groups often reunited but others remained separated until pupation. The transition from the natal to second leaf appears to be a crucial and dynamic time for larvae and events proceeded differently for most groups. During this period mothers moved actively among different leaves and branches in what seemed to be attempts to herd and reconstitute a single larval group. While we observed what we interpret as herding behavior in most species in this report, its possible importance to group safety and success remains open and in need of experimental study.Jacoby, 1880http://species-id.net/wiki/Proseicela_bicruciataProseicela bicruciata  during the first week of July 2011. A total of five females were found, each tending small groups of uniform larvae http://species-id.net/wiki/Proseicela_spectabilisProseicela spectabilis were taken by G.D. while walking the main trail leading to the Cascadas de San Rafael, Reventador, Napo province, Ecuador  on July 17, 2011. A single female  on 15 July 2011. Following discovery the larvae formed a small rosette with heads to the inside. The female and larvae were attached to the underside of a leaf of Cuatresia sp. (Solanaceae) (e larvae  was obseanaceae) . Two othPageBreakPageBreakProseicela species are known from understory Solanaceae at Cascadas de San Rafael, Ecuador. Several Proseicela antennalis adults  Bitter, 17 July 2011. As none of the individuals in these two species were reproducing, their interactions with offspring remain unknown, however high morphological similarity to adults of other Proseicela species in the area suggest they are good candidates to be subsocial. Platyphora amabilis  and Platyphora nigronotata  specimens were collected from Serra do Japi, Brazil where aspects of their biology has been studied  and Proseicela antennalis (7.5%), and between Proseicela spectabilis and Proseicela antennalis (9.0%) with all remaining pair-wise distances ranging between 10.2 and 18.6%. Thus all mean distances fall above the 2 to 6% range considered a threshold for distinct species, depending on COI evolution rates within particular clades  are now known and where the mode of reproduction has been recorded, all (17) are live-bearing or “larviparous” (Platyphora aulica and Platyphora sphaerica) have solitary larvae. Maternal care is now known to occur in six species and known not to occur in another eleven species. While five Proseicela species are maternal care providers, Proseicela crucigera in south-central Brazil has gregarious larvae but mothers do not extend care after larviposition .Aspects of the biology of 20 Neotropical species of iparous” . FurtherApocynaceae and Solanaceae associated taxa to one another and to other Doryphorini associated with eight other host plant families is best left to a multi-gene analysis . However, the COI data are sufficient to confirm the validity of 12 Solanaceae feeding species and supports the existence of two major clades, one of which contains non-maternal care providing species distributed primarily in the “cerrado” of central and southern Brazil. The second clade contains five maternal care species, three spePageBreakcies whose habits are unreported and one species, Platyphora aulica, which has apparently lost parental care. The latter is particularly interesting in that it is distributed over the northern portion of the Brazilian shield in “cerrado” habitats resembling those occupied by species in the non-maternal care clade. Further, our tree raises doubts about present generic concepts, at least for Solanaceae-feeding taxa. This problem is best seen in the small well-resolved clade containing Platyphora aulica, Platyphora amabilis and Proseicela vittata in Platyphora species , one solution may eventually consist of transferring most, or perhaps all, short-horned, Solanaceae-feeding Platyphora species into the genus, Proseicela.Because of the caveats to which mitochondrial data are subject , and limChrysomelinae are beginning to reveal patterns. The first is that maternal care behavior is primarily defensive in nature. Doryphora mothers straddle larvae and take measures to physically confront pedestrian predators. There is no convincing evidence that mothers act to supply or direct offspring to resources, although there are indications that Proseicela vittata mothers are modifying resources through vein-pinching. Proseicela and Platyphora mothers closely tend, straddle and herd offspring much as observed in Doryphora, but aggressive challenges of nearby insects or artificial stimuli have not been observed. For the most part, mothers remain immobile and close to their larvae , are likely to sequester only plant amyrins as precursors of saponin-defensive secretions but whether the secretions of either of these taxa are employed in defense of larvae remains unknown.As lkaloids . Adult PChrysomelinae are specialist feeders on one of approximately nine different host plant families, yet all maternal care species to date are restricted to only two, Apocynaceae and Solanaceae. Clearly more documentation is desirable, but if this pattern were to continue then it will be important to look for attributes possessed by these two plant families which promote evolution of maternal defenses but which are absent in other families, such as the Malpighiaceae, a family which hosts many poorly known Chrysomelinae species in South America.The second pattern beginning to emerge concerns choice of food plants. New World PageBreakGonioctena species occur from Europe , well-studied manipulators of host reproduction (oduction .Chrysomelinae and is not present in all species of these genera. PageBreakDoryphora species are exclusively associated with lianas in the family Apocynaceae, possess a long mesosternal horn, are oviparous and reproduce at the transition from dry to wet seasons. Proseicela and some species currently placed within Platyphora are associated with Solanaceae host plants, and are live-bearing throughout the year. While extreme rareness remains an impediment to the study of most Doryphora species, Proseicela and allied species on the eastern slope of the Andes in Ecuador can be found more predictably on moderately common host plants. Large voids remain in our understanding of the natural history of both groups, including the identity and importance of predators and parasitoids and the diverse ways in which mothers may be influencing the survival of offspring.Maternal care behavior appears limited to three genera of Neotropical


This study aims to observe the impact of the temperature of blood transfusion and infusion toward the perioperative cerebral oxygen metabolism and the postoperative cognitive recovery.n = 40) and control group (n = 40). The changes of nasopharyngeal temperature, middle cerebral artery blood flow, CERO2, and SjVO2 of the two groups were recorded at each time point for the assessment of the postoperative overall quality of recovery and cognitive recovery situation.Eighty patients of knee replacement under epidural and general anesthesia were randomly divided into warming blood transfusion and infusion (WBI) group (P < 0.05). The CERO2 values of the two groups at T3 were significantly higher than at T1, while the SjVO2 values were significantly decreased (P < 0.01).The nasopharyngeal temperatures of the two groups at different time points after transfusion were significantly lower than that at T1, and there was a significant difference between the two groups (The WBI can significantly reduce the occurrence of the perioperative hypothermia, while it has no significant effect toward cerebral oxygen metabolism, postoperative overall recovery, and recovery of cognitive function. Postoperative neuropsychological dysfunction is a common complication in elderly patients after joint replacement surgery. Some data indicated that  the inciLow temperature has been identified with positive effects toward cerebral protection both in animal experiments and clinical practice. Ohnishi et al.  studied n = 40) and control group (n = 40). WBI group used infusion fluid heating apparatus  to warm the infusion blood and fluid up to 37°C. In the control group, the infusion blood and liquid were kept at room temperature (24°C) for 15–20 min before the direct infusion without warming treatment. Except for the researchers, the subjects, the surgeons, and the serum biochemical parameter detectors did not know the patient's grouping situation. This study was conducted in accordance with the declaration of Helsinki. This study was conducted with approval from the Ethics Committee of General Hospital of Ningxia Medical University. Written informed consent was obtained from all participants.The study was a prospective, randomized, single-blind, and controlled trial, and 80 patients who undergone single knee replacement surgery from Mar. 2012 to Feb. 2013 in the Department of Osteology, General Hospital of Ningxia Medical University were enrolled. The patients aged 60–75 years old, without gender limitation. The patients would be excluded if they were with a preoperative history of cerebrovascular disease and external injuries; episode of thrombotic event; coagulation disorders; knee infection complicated by systemic infection; educational attainment <7 years; severe visual or hearing impairment, unable to cooperate the completion of cognitive function test; secondary emergency surgery; and when they refuse to sign the informed consent. The patients were randomly divided into WBI group (2), and continuous invasive arterial blood pressure , the patient had undergone L3-4 epidural catheterization, then intravenously administered with midazolam 0.05 mg/kg , fentanyl 2–3 μg/kg , propofol 2 mg/kg , and rocuronium 0.8–1.0 mg/kg  for the general anesthesia induction, with mechanical ventilation by endotracheal intubation. The anesthesia status was maintained with the inhalation of 0.5%–1.5% isoflurane, and propofol was continuously infused at 2–4 mg/kg/h with micropump, with intermittent boluses of fentanyl . The respiratory parameters were set as follows: tidal volume as 8–10 ml/kg, respiratory rate as 10 to 12 times/min, maintaining the end-tidal carbon dioxide (ETCO2) at 35–45 mmHg . The intraoperative inspired-oxygen concentration was 100%, and the intraoperative blood pressure fluctuation should be ensured not to exceed 20% of baseline values. The fluid infusion of all patients started with 500 ml compound sodium chloride, followed by the infusion demand at crystal/gel ratio 2:1 and at the speed of 5–8 ml · kg-1 · h-1. Lower extremity tourniquet was used during the surgery, with the inflation pressure at 350–400 mmHg. The patients of the two groups were infused allogeneic or preoperative autologous blood through the peripheral vein 10 min before removing the tourniquet, with the speed as 15 ml/min. During the surgery, the amount of blood loss was calculated by the aspirator volume and gauze weighing. The postoperative wound drainage was recorded hourly; the drainage device could be removed until 48 h after the surgery. The postoperative patient-controlled epidural analgesia was performed, with 0.2% ropivacaine  2 ml/h for 72 h, followed by oral non-steroidal anti-inflammatory analgesics. The surgeons and the anesthesiologists of all patients were the same group of people.All patients were given epidural and general anesthesia. Before anesthesia, the patient was intravenously injected with 10 mg dexamethasone and 0.5 mg atropine; the patient who had rapid heart rate would be given 0.3 mg scopolamine instead of atropine. Under the monitoring of electrocardiogram, saturation of pulse oxygen (SPO2) and cerebral extraction rate of oxygen (CERO2) monitoring: the patient was in supine position, with head in neutral line and parallel to the bedside. After local anesthesia, the internal jugular vein paracentesis was performed from the spot slightly outside the carotid pulse and parallel to the lower edge of thyroid cartilage, and along the ipsilateral external ear direction. After the success of paracentesis, 5F bi-cavity deep vein catheter was directed inside, with the catheter tip in the equivalent position of external ear canal . The catheter was sealed with heparin saline, and the radial arterial blood and jugular bulbar blood were extracted synchronously at T0–T5 for the blood gas analysis, respectively; the SjVO2 value was monitored and CERO2 calculated. (4) Assessment of the postoperative recovery quality and the cognitive function: the Post-operative Quality Recovery Scale (PQRS) of all subjects were tested preoperatively, 15 and 40 min after extubation, postoperative 1 and 3 days, respectively . (3) Jugular venous oxygen saturation , but the difference between the two groups at all time points was not statistically significant (P > 0.05). The RI of control group at T2 was significantly higher than at T1 (P < 0.05), while the difference between the two groups at different time points was not statistically significant . However, the CERO2 and SjVO2 at each time point had no statistically significant difference between the two groups  could cause chills, coagulation disorders, cardiovascular dysfunction, metabolic abnormalities, etc. ,16. Low In this study, transcranial Doppler instrument was used to monitor the indicators which could reflect the cerebral blood flow velocity (CBFV) in the middle cerebral artery, including Vs, Vd, and Vm (mean blood flow velocity), among which Vs represented the highest blood flow velocity in systolic period, reflecting the highest MCA flow velocity during entire cardiac cycle; Vd represented the highest MCA blood flow velocity at the end of cardiac cycle, reflecting the vascular resistance; RI mainly reflected the cerebrovascular resistance condition. If there was no significant change in vessel diameter, the above indicators would increase with the CBFV, and the cerebral blood flow (CBF) would also increase; but when the vascular spasm or strong contraction happened, through CBFV would increase, CBF might have no change or even decrease; while the vasodilation at this time would make CBFV reduce, CBF would increase or exhibit no change. In this study, the Vs of the two groups decreased immediately after the transfusion and still decreased 30 min after the transfusion; it might probably be because of the relief of tourniquet pressure, the lower limb blood vessels could dilate, causing the CBF slow down. The intergroup comparison exhibited no significant difference, indicating that compared with the control group, the WBI had no effect on cerebral blood flow velocity of elderly knee replacement patients; it might partially be because the transfusion volume in this study was relatively small and the degree of hypothermia did not break 35°C.2 and SjVO2 were simultaneously monitored for the comprehension of the balance of perioperative cerebral oxygen supply and demand. Under normal circumstances, when the CBF is constant, CERO2 would reduce as the temperature decreases, while SjVO2 would relatively increase. In this study, the CERO2 of the two groups 30 min after transfusion were significantly higher than those before the transfusion, and SjVO2 were significantly lower than those after anesthesia while before transfusion, but they were still within the normal ranges. We considered the above situation might be because of the insufficient cerebral blood supply, which was induced by the acute bleeding in short time after the tourniquet was released. Because the major organ reservation and the compensatory function of elderly patients significantly reduce, the biotransformation and elimination rates of liver and kidney toward the narcotic drug would significantly prolong, resulting in the poor quality of postoperative recovery, even the occurrence of POCD [In this study, CERO of POCD . Althoug of POCD  indicateAlthough studies have shown  that theThe objects of study are patients with knee replacement surgery, and the intraoperative blood transfusion amount is less than normal. In addition, the follow-up of postoperative overall recovery quality and cognitive function restoration is performed just for three postoperative days, without long-term follow-up (6 months or 1 year). These are the limitations of this study. Next, based on results of this study, the research objects should be patients with large blood transfusion in surgery. The change of perioperative cerebral oxygen metabolism and overall recovery quality and cognitive function restoration in one postoperative year will be observed. The objective is to decide performing WBI or not, and guide clinical reasonable blood transfusion.It is found that the blood transfusion is a risk factor for postoperative early cognitive dysfunction in elderly patients. Whether intraoperative infusion of preoperative autologous blood or allogeneic blood will influence the postoperative cognitive function to a certain degree, and this mainly depends on the amount of infused blood. More than 1,000 ml of infused blood is considered as the independent risk factor for postoperative delirium . In thisFor elderly patients treated by single knee replacement surgery, intraoperative WBI can reduce the occurrence of perioperative hypothermia, which is not contrary to the concept of cerebral protection by low temperature. The intraoperative WBI has no significant effect on the perioperative cerebral oxygen metabolism or cognitive function recovery. In addition, sometimes the WBI device cannot be used, for saving medical resources and reducing expense of patients and medical burden of society.POCD: postoperative cognitive dysfunction; RI: resistance index.The authors declare that they have no competing interest.XN and CW designed this paper and performed critical revision of the manuscript. YY and YC performed data collection. YW analyzed the date. XN wrote the manuscript. All authors read and approved the final manuscript.


Artemia franciscana, transporting cysts (diapausing eggs), and favouring rapid range expansions from introduction sites. Here we address the impact of bird migratory flyways on the population genetic structure and phylogeography of A. franciscana in its native range in the Americas. We examined sequence variation for two mitochondrial gene fragments (COI and 16S for a subset of the data) in a large set of population samples representing the entire native range of A. franciscana. Furthermore, we performed Mantel tests and redundancy analyses (RDA) to test the role of flyways, geography and human introductions on the phylogeography and population genetic structure at a continental scale. A. franciscana mitochondrial DNA was very diverse, with two main clades, largely corresponding to Pacific and Atlantic populations, mirroring American bird flyways. There was a high degree of regional endemism, with populations subdivided into at least 12 divergent, geographically restricted and largely allopatric mitochondrial lineages, and high levels of population structure (ΦST of 0.92), indicating low ongoing gene flow. We found evidence of human-mediated introductions in nine out of 39 populations analysed. Once these populations were removed, Mantel tests revealed a strong association between genetic variation and geographic distance . RDA showed that shared bird flyways explained around 20% of the variance in genetic distance between populations and this was highly significant, once geographic distance was controlled for. The variance explained increased to 30% when the factor human introduction was included in the model. Our findings suggest that bird-mediated transport of brine shrimp propagules does not result in substantial ongoing gene flow; instead, it had a significant historical role on the current species phylogeography, facilitating the colonisation of new aquatic environments as they become available along their main migratory flyways.Since Darwin’s time, waterbirds have been considered an important vector for the dispersal of continental aquatic invertebrates. Bird movements have facilitated the worldwide invasion of the American brine shrimp  Since Given that continental aquatic invertebrates are unlikely to be in migration-drift equilibrium , recent Artemia franciscana (Crustacea: Anostraca), the most widely distributed brine shrimp in America, occurs in hypersaline habitats from Canada to Chile and many Atlantic islands , confirms shorebirds as an effective agent of dispersal in North America  its distribution encompasses three continental-scale bird migratory flyways spanning both North and South America , but is highly fragmented due to its habitat requirements , (2) its habitats are frequented by migratory shorebirds; Artemia is an important prey of these and other waterbirds and its cysts can be readily quantified in waterbird excreta A. franciscana throughout its known native range  using sequence variation for two mitochondrial genes (COI and 16S). Our results indicate a high level of genetic structure and endemism at a continental scale, identify the impact of human introductions and suggest a direct link between bird migratory routes  and the historical colonization of A. franciscana throughout the Americas, revealing a key role for birds in initial founder events.Here, we carry out the first comprehensive phylogeographic study of A. franciscana populations across its American geographical distribution, from Canada to Chile and Argentina, including Caribbean islands and a population from Cape Verde (We obtained samples from 39 pe Verde . Most saArtemia-specific primers 1/2COI_Fol-F and 1/2COI_Fol-R (c Oxidase Subunit I gene (COI). We also amplified and sequenced a 535 bp fragment of the 16S ribosomal RNA gene for a subset of individuals carrying different COI haplotypes to facilitate comparison with other published sequences using primers 16Sar-5′/16Sbr-3′  and 0.5 µM of each primer. PCR conditions were as follows: 94°C for 3 min, followed by 35 cycles of 45 s at 94°C, 60 s at 45°C (60–64°C for 16S locus), and 60 s at 72°C, followed by 5 min at 72°C. PCR products were purified for sequencing using ExoSAP-IT® , cleaned with Sephadex®-G50 , and labelled using the BigDye Terminator Sequencing Ready Reaction v3.1 kit (Applied Biosystems). The resulting fragments were separated on an ABI 3130xl genetic analyzer. Sequences were checked, edited, and aligned using Sequencher® v4.5 . All sequences were deposited in GenBank [accession numbers KF662951–KF663043 and KF725843–KF725869]. Available published sequences of the same gene fragments, to which we could assign a geographic origin, were also included in our phylogenetic analyses .DNA extractions were carried out on individual cysts (previously rinsed in distilled water), whole nauplii or partial adults using a HotSHOT protocol . We usedOI_Fol-R  to ampli16Sbr-3′ . PCRs wehttp://www.phylo.org), optimising free model parameters and executing 1000 rapid bootstraps. Average genetic distances between the main COI lineages – corrected by the K2P + G substitution model – were carried out using MEGA5. Additionally, to identify lineages in our COI dataset, we used the Automatic Barcode Gap Discovery (ABGD) approach (http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html).Neighbour Joining (NJ) and Maximum Likelihood (ML) phylogenetic trees were inferred for both COI and 16S gene fragments. NJ trees were constructed using evolutionary distances computed using the Maximum Composite Likelihood method and 1000 bootstrap replicate tests in MEGA5 . The besapproach  using thHs (standardized haplotype diversity) for COI was computed using the program RAREFAC . A geographic distance matrix was then computed using Geographic Distance Matrix Generator v.1.2.3 . We used a population geographic distance matrix  was tested using Mantel tests on IBDWS v.3.21 . Prior te matrix  and a poST values using Kimura 2-Parameter as the evolutionary model for COI data. Environmental variables were whether the flyway overlapped with the Artemia population  and introduction history (0 or 1). We modelled spatial variables using latitude and longitude (x and y).We used canonical Redundancy Analysis (RDA) in CANOCO  to estimhttp://www.birdlife.org/datazone/userfiles/file/sowb/flyways/). These data were used as a proxy for bird movements between locations. This approach is likely to be a rough approximation to the probability of bird migration between locations, given that more precise estimates cannot be obtained due to the absence of sufficient shorebird ringing data for the whole of the Americas. Even if extensive ringing data was available, this would only estimate current bird movements, whereas climate changes since the Pleistocene are likely to have strongly affected bird movements (We used the overlap of sampled populations with the three main migratory flyways in America based on two sources (1) ovements .http://dx.doi.org/10.5061/dryad.7kb11).RDA analyses were carried out using each set of migratory flyway data  as suggested by the program, to 1.0 and used Kimura 80, identifying 12 groups with a prior intraspecific distance of = 0.0028 . When the prior intraspecific distance increases to 0.0046 (not high by any standards) then the number of partitions reduces again to 1.Introduced populations below). In addition, lineage 1 contained six closely related haplotypes only found in Mono Lake – which harbours a population often considered as a separate species, A. monica – and haplotypes found in three Colombian populations , five Mexican populations, and the Jamaican population. Two other lineages were distributed across the continental E-W divide, creating contact zones between lineages. In lineage 12, most of the haplotypes were found in Argentinean populations, but four of these haplotypes (three of them shared across populations) were found in a Chilean Altiplano population . Lineage 11, mainly Caribbean, has a few haplotypes in two Mexican populations from the Pacific side . Finally, although both lineages 1 and 11 are found in Colombia, they were not found together in any of the populations sampled. The genetic divergence between the 12 main lineages ranged from 1.8% (between lineages 1 and 4) to 6.0% (between lineages 2 and 9)  and the other split between two sub-branches along the Pacific Rim (lineages 1–7) and in Central Canada (lineage 8). Overall, there were at least 12 mtDNA lineages, most of them well supported . With th2 and 9) .A. franciscana populations. Interestingly, 16S haplotypes from Jesse Lake (Nebraska) belong to the Central Canadian lineage (lineage 8) together with Little Manitou, Muskiki, Meacham and Chaplin haplotypes.The 16S alignment contained 408 bp from 122 individuals, which collapsed into 59 haplotypes. There were two singleton indels, 63 variable sites and 43 parsimony informative sites. In contrast to the COI analysis, the NJ and ML reconstructions were poorly resolved, especially the basal branches, but most of the lineages recovered by the COI analyses were also recovered for the 16S data, with variable levels of support see . COI linH, and details of the haplotypes present in each population). The number of haplotypes per population ranged from one to seven. The highest standardized gene diversity (Hs) was found in the Mexican population MexH, whereas five populations  were fixed for a single haplotype. Most haplotypes were found within single countries, except for several haplotypes shared between some countries and the two commercial USA populations SFB and GSL , with ΦST being highly significant between all populations except for three lakes from Central Canada, plus one pair from Chile/Argentina .Mantel tests on pairwise genetic and geographic distances for populations ranging from Chile-Argentina to Canada, excluding those inferred to be introduced, revealed a strong IBD pattern  with a hp < 0.02 for all the correlations with genetic distance, whether or not geographical distance was controlled for). The effect of using different flyway data was quite small. Flyway explained 18.7–21.2% of variance in population genetic distance, depending on the flyway dataset used  division reflecting the recognized zoogeographic regions and the long isolation of the continents  extending into new breeding areas becoming available in the north and new wintering sites in the southern extreme of the continent  ntinents  – strongdent see , althougontinent . These rterbirds  and the terbirds . Althougterbirds , the hypA. franciscana agree with other studies in North America pointing to an effect of bird movements on the genetic structure of passively dispersed aquatic invertebrates , and those for A. salina in its native range  the time frame of their fragmentation can be approximated roughly using a COI molecular clock for other shrimp taxa , for which evidence of nuclear DNA (nDNA) introgression among lineages exists . FurtherNatural spread of lineages from refugial areas is likely to result in contact zones between lineages, which we expect to be sharp, as we found between lineages 1 and 11 in Colombia, where despite two lineages being present in the area, there are no sites where both co-occur.A. franciscana, some of them, like lineages 9 (Puerto Rico) plus 10 (Cape Verde) compared to all the others, or lineages 11 (Circum Caribbean) plus 12 (Argentina-Chile) compared to all the others, have genetic divergences of over 5%. Surprisingly, reproductive isolation – due to ecological specialisation and local adaptation – has only been reported between Mono Lake and San Francisco Bay populations  and distant populations as a criterion of recent human mediated introduction. Using this criterion, we identified nine populations where genetic evidence pointed to putative human introductions into Mexico, Cuba, Jamaica, Brazil and Chile. For four of these populations, the occurrence of intentional introductions in the same or nearby sites during the 1970s could be documented. Therefore, our genetic data confirms that the established A. franciscana populations in these locations are, at least partially, of introduced origin, and validates our criterion. As for the impact of introductions on native populations, four Mexican populations contained additional private haplotypes not closely related to the introduced ones, which suggests the presence of a pre-existing population before the introduction and the likely introgression of both populations with persistence of native haplotypes, although nuclear loci would be necessary to confirm this. In two populations, we found a discrepancy between our genetic results and previously published ones. The first case is the MEXLC population, which we regard as introduced (lineage 1), whereas in A. franciscana is known to hybridize even with the genetically divergent A. persimilis (Daphnia) from populations where commercialised strains were introduced.Given the strong phylogeographic structure of rsimilis . This farsimilis . DespiteThe high genetic richness found in our COI analyses, and the presence of private haplotypes belonging to lineage 1 in distant populations away from SFB and GSL, including three Colombian and five Mexican populations likely to be native, indicates that the natural distribution of lineage 1 extended further than the two commercialized populations before any human introductions took place. This was also in evidence from our 16S analyses, which included more extended geographic coverage of NW America, and revealed further sites within lineage 1 in the W USA and British Columbia harbouring private haplotypes not found in SFB or GSL. This is also consistent with the presence of private and closely related haplotypes at Mono Lake. The peculiarity and fragmentation of the habitats used by the species, the potential of salinity and varying ionic composition to act as a strong selective agent, the capacity to produce massive quantities of resting eggs that can be readily dispersed by birds, combined with the apparent limitation in modern gene flow, makes this group an ideal system for further studies testing the role of local adaptation and mass effects on reducing gene flow between populations.A. franciscana across the world, and the high genetic and ecological richness of its native populations, further population translocations should be highly discouraged, and the use of native strains as a source of cysts should be encouraged even within the Americas.Given the impact of the invasive A. franciscana phylogeography in its native range was shaped by (1) Pliocene/Pleistocene climate fluctuations, which contributed to changes in the areas available to the species, (2) historical bird-mediated colonization along migratory flyways, which shaped the East-West population division, (3) strong and persistent founder events, facilitated by high population growth rates and large population sizes, preventing further gene flow despite ongoing bird-mediated dispersal, and (4) human introductions coupled with regional bird dispersal, explaining the large but localised geographic range of the lineages derived from the commercially exploited North American populations. Our findings suggest that, at a continental scale, bird-mediated transport of invertebrate propagules does not result in substantial ongoing gene flow, but instead determines species phylogeography, facilitating the colonisation of newly available aquatic environments along bird flyways.Our analyses suggest that 10.7717/peerj.200/supp-1Table S1Click here for additional data file.10.7717/peerj.200/supp-2Table S2Click here for additional data file.


Wistar rats by an intraperitoneal injection of streptozotocin. Retinal ischemia was induced by increasing intraocular pressure to 120 mm Hg for 5 min; this maneuver started 3 days after streptozotocin injection and was weekly repeated in one eye, while the contralateral eye was submitted to a sham procedure. The application of ischemia pulses prevented a deficit in the anterograde transport from the retina to the superior colliculus, as well as an increase in astrocyte reactivity, ultraestructural myelin alterations, and altered morphology of oligodendrocyte lineage in the optic nerve distal portion at early stages of experimental diabetes. Ischemia tolerance prevented a significant decrease of retinal glutamine synthetase activity induced by diabetes. These results suggest that early vision loss in diabetes could be abated by ischemic conditioning which preserved axonal function and structure.Diabetic retinopathy is a leading cause of blindness. Visual function disorders have been demonstrated in diabetics even before the onset of retinopathy. At early stages of experimental diabetes, axoglial alterations occur at the distal portion of the optic nerve. Although ischemic conditioning can protect neurons and synaptic terminals against ischemic damage, there is no information on its ability to protect axons. We analyzed the effect of ischemic conditioning on the early axoglial alterations in the distal portion of the optic nerve induced by experimental diabetes. Diabetes was induced in  Diabetic retinopathy (DR) is one of the most common and feared complications of diabetes. This disease is a leading cause of blindness in people of working age in industrialized countries and affects the daily lives of millions of people. Vision loss mainly occurs due to chronic hyperglycemia, vascular damage and leakage, edema formation, capillary basement membrane thickening, neovascularization, hemorrhage, and ischemia Although retinal microcirculatory abnormalities and neurodegeneration could explain some of the visual deficit induced by diabetes, visual function disorders have been demonstrated in diabetics with very early retinopathy or even before the onset of retinopathy. Psychophysical methods and electrophysiologic measurements indicate abnormalities in visual pathway function per se, and trigger yet incompletely described mechanisms that result in retinal tolerance to the damaging ischemic event. Besides the effect of ischemic conditioning against an acute ischemic episode, brief retinal ischemia pulses significantly prevent retinal functional, morphological, and vascular alterations induced by experimental diabetes Retinal chronic ischemia is one of the DR hallmarks. At present, there is no effective treatment against retinal ischemic injury. However, it is possible to activate an endogenous protection mechanism that protects from retinal ischemic damage by ischemic pre-conditioning (IPC) stratum zonale (SZ) and the stratum griseum superficiale (SGS), the most superficial layers of the SC. In control animals, intense CTB-stained retinal projections were observed in the SC  and the mean axon area in the distal ON portion . The appTo investigate the effect of ischemic tolerance on diabetes-induced astrocyte alterations in the distal portion of the ON, GFAP-immunoreactivity was analyzed in diabetic rats submitted to ischemia pulses in one eye, and a sham procedure in the contralateral eye. A significant increase in GFAP-immunoreactivity in the transverse ON portion was observed in diabetic eyes submitted to a sham procedure as compared with age-matched controls, whereas ischemic tolerance decreased this parameter . Figure P<0.01) and diabetic eyes subjected to ischemia pulses (P<0.01). The plot slope in diabetic ONs from eyes submitted to ischemia pulses did not differ from that obtained in control (non-diabetic) ONs. At ultrastructural level, compact bundles of myelinated axons fascicles were observed in control ONs . In the In order to evaluate the effect of ischemic tolerance on OL-linage, the effect of ischemia pulses on the population of immature OL (O1+) and OL precursor (PDGFRα) cells in diabetic eyes was analyzed. In the diabetic distal ON portion, an increase in O1- and PDGFRα-immunoreactivity, with hypertrophic somas and a high number of processes were found . The appIn order to analyze the mechanism involved in the protection induced by ischemic conditioning on the diabetic optic pathway, retinal GS activity was assessed at 6 weeks of diabetes onset. In diabetic eyes submitted to a sham procedure, a significant decrease in GS activity as compared with control retinas was observed, whereas ischemia pulses significantly prevented the decrease in this parameter .per se, prevented a deficit in the anterograde transport from the retina to the SC, and the decrease in axon number and myelination, as well as the alterations in astrocytes and OL lineage in the distal portion of the ON induced by experimental diabetes.The present results indicate that ischemic conditioning, which showed no effect Evidence of axon degeneration occurring separate from, and before soma degeneration has been described in many neurodegenerative diseases, such as Alzheimer disease, Parkinson disease, and glaucoma Several lines of evidence strongly support that ischemic tolerance induced by IPC or PostC is an important phenomenon of the central nervous system adaptation to subsequent or previous ischemia, respectively. In fact, ischemic conditioning is actually considered the strongest known procedure to prevent or avoid neurodegeneration. Ischemic tolerance works specifically in vulnerable neuronal populations, such as pyramidal neurons in the hippocampal CA1 region As shown herein, ischemic conditioning prevented the deficit in the CTB anterograde transport from the retina to the SC, its primary projection site, indicating that ischemic pulses prevented a “misconnection” between the retina and the SC induced by experimental diabetes. In animals that had been diabetic for 6 weeks, although no obvious changes in retinal morphology and in the number of RGCs were observed, a large increase in astrocyte reactivity (as shown by GFAP-immunoreactivity) occurred in the distal portion of the ON, which coincided with a significant axon loss, being large axons significantly more vulnerable than small axons. The application of ischemia pulses prevented the increase in GFAP-immunoreactivity, and the decrease in axon number and mean axonal area.It is well established that the first site of neuronal damage may be remote from the site of insult 29]. For example, in transected motor axons, neuromuscular junctions that are many centimeters from the lesion degenerate first, while the axons immediately adjacent to the lesion remain intact for two to three times longer than the distant terminals Glutamate is the main excitatory neurotransmitter in the mammalian retina, but it is neurotoxic when present in excessive amounts. Müller cells surround glutamatergic synapses, and express glutamate transporters, and the glutamate metabolizing enzyme, GS. Extracellular glutamate is transported into Müller cells and amidated by GS to the non-toxic amino acid glutamine, which is then released by Müller cells and taken up by neurons, where it is hydrolyzed by glutaminase to form glutamate again. Thus, Müller cells play a substantial role in the retinal glutamate/glutamine cycle activity, and their role in maintaining low extracellular concentrations of glutamate may be particularly critical in diabetes The past two decades have provided interesting insights into the mechanisms and potential applications of ischemic tolerance which can protect neurons and improve neuronal survival after ischemia or even against several types of non-ischemic damage. These results suggest that early vision loss in diabetes could be abated by ischemic conditioning which preserved distal axonal function and structure before the neuronal soma loss. Moreover, the present results add new potentialities to the therapeutic effects of ischemic tolerance, which is axon protection. Thus, ischemic tolerance could have promise for application in other neurodegenerative axonal diseases.All animal procedures were in strict accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The ethic committee of the School of Medicine, University of Buenos Aires ) approved this study.Wistar rats  were housed in a standard animal room with food and water ad libitum under controlled conditions of humidity and temperature , and under a 12-hour light/12-hour dark lighting schedule (lights on at 8∶00 AM).Male For diabetes induction, a single dose of STZ , 60 mg/kg , was i.p. injected. Control rats received an equal volume of citrate buffer. The animals were examined 72 hours after injection using a glucose meter , and those with blood glucose levels greater than 350 mg/dL were considered diabetic. Body weight and plasma glucose level were determined weekly. For the assessment of body weight and blood glucose levels, a group of diabetic animals with intact eyes was included.Retinal ischemia was induced as previously described Rats were anesthetized nd a drop of proparacaine (0.5%) was topically administered for local anesthesia. Four microliters of a solution of 0.2% cholera toxin β-subunit (CTB) conjugated to Alexa 488 dye  in 0.1 M PBS (pH 7.4) was injected into the vitreous using a 30-gauge Hamilton syringe . Injections were applied at 1 mm of the limbus and the needle was left into the eye for 1 minute to prevent volume loss. At 3 days after injection, rats were anesthetized and intracardially perfused with saline solution followed by a solution containing 4% formaldehyde in 0.1 M PBS (pH 7.4). Their brains were carefully removed, post-fixed overnight at 4°C, and immersed in a graded series of sucrose solutions ; coronal sections (40 µm) were obtained using a freezing microtome . Nuclei were stained with fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI), mounted with antifade medium  and viewed with a fluorescence microscope  connected to a video camera  attached to a computer running image analysis software . For immunofluorescence studies, comparative digital images from different samples were grabbed using identical exposition time. Digitalized captured TIF-images were finally assembled and processed in Adobe Photoshop SC . For illustration purposes, the image quality was optimized by making adjustments in brightness and contrast that were constantly applied to all images. No other adjustment was made. All the nomenclature used in the paper follows that of Paxinos and Franklin + pixels. Finally, a colorimetric thermal representation was applied (from 0% = blue to 100% = red). The number of sections and the thickness (2x) were used for a final reconstruction of the retinal projection to the SC. Five animals per group were used.CTB staining was quantified as previously described Anesthetized rats were intracardiacally perfused as previously described herein. Then, eyes and brains were carefully removed and immersed for 24 h in the same fixative. Eyes were embedded in paraffin and sectioned (5 µm) along the vertical meridian through the ON head. Sections were stained with hematoxylin and eosin and used for morphometric analysis. Digital images were obtained at 1 mm dorsal and ventral from the optic disk. The total retinal, inner plexiform layer, inner nuclear layer and outer nuclear layer thickness (in µm) was measured for each eye. For each eye, results obtained from four separate sections were averaged and the mean of 10 eyes was recorded as the representative value for each group. For Brn3a immunostaining, retinas were isolated and flat-mounted with the vitreous side up in glass slides.ON portions at 2 mm before the optic chiasm  were dissected out and after several washings, tissue blocks were postfixed in 2% osmium tetroxide in sodium phosphate buffer for 1 hour. Dehydration was accomplished by gradual ethanol series, and tissue samples were embedded in epoxy resin. Semithin sections (1 µm) were obtained with an ultramicrotome, stained with toluidine blue and used for morphometric analysis. In some cases, the ONs were dehydrated and embedded in paraffin. Transverse and longitudinal sections (7 µm) were obtained and used for immunostaining analysis. ON total area was calculated from light microphotographs of transverse semithin sections obtained with a final magnification of 20x. The nerve was divided in four quadrants and the number and area of axons were calculated in each section (in areas randomly chosen in each quadrant) from micrographs obtained at 100x. Images were converted to 8-bits grey scale and a manual threshold value, first determined by visual examination, was constantly applied. Finally, images were converted to a binary form. Myelinated fiber sizes were digitally delineated by the outer margin of their myelin sheaths and axon areas were measured. For each ON, results obtained from four separate sections were averaged, and the mean axon number and area of six nerves was recorded as the representative value for each group.The brains were coronally sectioned (behind the optic chiasm) and the anterior portion was included in paraffin as previously described Sections were immersed in 0.1% Triton X-100 in 0.1 M PBS for 10 min. Antigen retrieval was performed in paraffin sections, by heating (90°C) slices for 30 minutes in citrate buffer (pH 6.3). Sections were pre-incubated with 5% normal horse serum for 1 h, and then incubated overnight at 4°C with the following primary antibodies: a mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody conjugated to Cy3 , a rabbit polyclonal anti-myelin basic protein (MBP) antibody, a mouse polyclonal IgM O1 antibody ), a goat polyclonal anti-platelet-derived growth factor receptor-α (PDGFR-α) antibody . After several washings, a donkey anti-mouse IgM secondary antibody conjugated to Cy3 , and a donkey anti-Goat secondary antibody conjugated to DyLight 488  and a goat anti-rabbit secondary antibody conjugated to Cy2  were added, and sections were incubated for 2 h at room temperature. Regularly, some sections were treated without primary antibodies to confirm specificity. After immunostaining, nuclei were stained with DAPI, mounted with antifade medium and viewed with a fluorescence microscope as described.2. For each group, results obtained from four separate quadrants were averaged, and the mean of 5 eyes was recorded as the representative value.For all morphometric image processing and analysis, digitalized captured TIF-images were transferred to ImageJ software . For immunostaining density analysis and area determination  in ON sections, images were converted to 8-bits grey scale and in each case, a manual threshold value was determined to binarize the images. Results obtained from three sections were averaged and recorded as the representative value for each ON. Six animals per group were used. For RGCs density determination, the retina was divided in 4 quadrants, and images from the central and peripheral area were obtained. The number of Brn3a(+) cells was measured and expressed as number of cells/mmAfter anesthesia and thoracotomy, animals were perfused through the left ventricle with a fixative solution containing 2% glutaraldehyde and 4% formaldehyde in 0.1 M PBS (pH 7.4). Semithin sections were obtained as described above and ultrathin sections were stained with uranyl acetate and lead citrate. Finally, sections were viewed and photographed using a Zeiss EM 10 C transmission electron microscope. Electron microscopical photographs were obtained with a final magnification of 3000 times. Axon areas were calculated from approximately 10 photographs (25×25 µm) per nerve. The calculation of axon-myelin ratio was obtained from no fewer than 200 myelinated axons per nerve. Data from four nerves were averaged and recorded as the representative slope value for each group.2, 50 mM β-mercaptoethanol, 20 mM ATP, 100 mM glutamate, and 200 mM hydroxylamine, adjusted to pH 7.2). Tubes were incubated for 15 min at 37°C. The reaction was stopped by adding 0.6 ml of ferric chloride reagent . Samples were placed for 5 min on ice. Precipitated proteins were removed by centrifugation, and the absorbance of the supernatants was read at 535 nm against a reagent blank. Under these conditions, 1 µmol of γ-glutamylhydroxamic acids gave an absorbance of 0.340. GS specific activity was expressed as µmoles of γ-glutamylhydroxamate per hour per milligram of protein. Data from eight retinas were averaged and recorded as the representative value for each group.Each retina was homogenized in 200 µl of 10 mM potassium phosphate, pH 7.2. Glutamine synthetase (GS) activity was assessed as described Statistical analysis of results was made by a two-way analysis of variance (ANOVA) followed by Tukeýs test.


Simple sequence repeat (SSR) markers are widely used tools for inferences about genetic diversity, phylogeography and spatial genetic structure. Their applications assume that variation among alleles is essentially caused by an expansion or contraction of the number of repeats and that, accessorily, mutations in the target sequences follow the stepwise mutation model (SMM). Generally speaking, PCR amplicon sizes are used as direct indicators of the number of SSR repeats composing an allele with the data analysis either ignoring the extent of allele size differences or assuming that there is a direct correlation between differences in amplicon size and evolutionary distance. However, without precisely knowing the kind and distribution of polymorphism within an allele (SSR and the associated flanking region (FR) sequences), it is hard to say what kind of evolutionary message is conveyed by such a synthetic descriptor of polymorphism as DNA amplicon size. In this study, we sequenced several SSR alleles in multiple populations of three divergent tree genera and disentangled the types of polymorphisms contained in each portion of the DNA amplicon containing an SSR. The patterns of diversity provided by amplicon size variation, SSR variation itself, insertions/deletions (indels), and single nucleotide polymorphisms (SNPs) observed in the FRs were compared. Amplicon size variation largely reflected SSR repeat number. The amount of variation was as large in FRs as in the SSR itself. The former contributed significantly to the phylogenetic information and sometimes was the main source of differentiation among individuals and populations contained by FR and SSR regions of SSR markers. The presence of mutations occurring at different rates within a marker’s sequence offers the opportunity to analyse evolutionary events occurring on various timescales, but at the same time calls for caution in the interpretation of SSR marker data when the distribution of within-locus polymorphism is not known. Simple sequence repeats  Citrus data represent sequences of individual trees each belonging to a different species, variety or cultivar; the Jacaranda data were collected in four different sampling sites considered as four distinct populations; and the oak data came from a single mixed stand of sessile/pedunculate oaks . The Citrus, Jacaranda, and Quercus data sets were submitted to partially different types of analyses according to the structure of each data set.Three data sets were utilized in this study. The structures of the three data sets analysed here varied in the following ways: the Citrus species being derived by natural hybridization from ancestral species, alleles were chosen to minimize known hybrids from the data set of Barkley et al. Citrus accessions. The individual identity of each accession  is given in the Supplementary Jacaranda data were obtained from three sites in French Guiana  and one in Brazil (Tapajos)  . The QueJacaranda markers were obtained following the method developed for SSR markers in tropical crops by Billotte et al. RsaI, followed by an enrichment step in (GT)n and (GA)n repeats by hybridization of cleaved fragments with biotin-labelled (CA)8 and (CT)8 oligoprobes and capture of the selected sequences with streptavidincoated magnetic beads. Those fragments were ligated into a pGEM-TEasy vector , and plasmids were then used to transform competent XL-1 Blue strain Escherichia coli . Positive colonies were tested using colony polymerase chain reaction (PCR) to check the presence of inserts. Amplified fragments were then transferred from an agarose gel onto a N+ Hybond membrane for SSR presence screening by hybridization with labeled γ−32 P (GT)15 and (GA)15 oligoprobes. One hundred and thirteen bacterial clones containing plasmids with inserts that gave strong hybridization signals, with sizes ranging from 500 to 1000bp, were selected and cultured. Plasmids were extracted and sequenced using the universal T7 primer on an automated ABI 377 capillary sequencer . After discarding duplicates, hybrid clones and clones with the SSR region too close to either end of the sequence, thirteen sequences were suitable for primer design and allowed a successful design using Oligo 3 software . Upon screening for polymorphism and clarity of PCR patterns, three primer pairs were chosen for subsequent analyses. Primer sequences, annealing temperatures and GenBank/EMBL accession numbers are shown in Taq buffer, 0.26 mM of dNTP, 0.03 U/µL Taq DNA polymerase  and 0.54 µM of each primer . For Jc3A10 primers, 0.3% BSA was added. An initial denaturation at 94°C for 5 min was followed by 35 cycles of 94°C for 30 s, annealing temperature for 30 s and 72°C for 30 s, and a final extension at 72°C for 10 min. Genotyping was performed using fluorescently labeled primers  in the previous PCR protocol and fragments were separated on an ABI 3130XL capillary sequencer  using ABI POP4 and Applied Biosystems LIZ-500 as internal standard, following the manufacturer’s instructions. DNA was bidirectionally sequenced directly from PCR amplification products. When necessary, gametic phase was determined in heterozygotes by cloning amplicons and sequencing at least one allele per individual following the TA cloning kit protocol from Invitrogen . PCR products were ligated with the plasmid pCR®2.1 and used to transform competent DH5 cells. After an overnight incubation at 37°C, white colonies were isolated. The presence of an SSR allele was checked by PCR with universal M13 primers. PCRs consisted of 1 µL of cultivated colonies, 1× Taq buffer, 0.125 mM dNTPs, 0.025 U/µL Taq DNA polymerase  and 0.83 µM forward and reverse primers. Cycling conditions consisted of 94°C for 5 min; 35 cycles of: 94°C for 1 min, 50°C for 1 min, 72°C for 2 min; and one cycle of 72°C for 5 min. PCR products were checked on a 4% agarose gel in 0.5× TAE. Plasmids were sequenced with the ABI BigDye® Terminator V3.1 kit  following the manufacturer’s protocol. Cloning products were diluted 1∶40, purified with ExoSap-IT , and separated on an ABI 3130XL capillary sequencer . All sequences were aligned and edited using CodonCode Aligner V1.6.3 .Citrus, Jacaranda, and Quercus), each allele was characterised by the following information: (a) amplicon size variation, (b) SSR variation, (c) FR sequence variation and (d) amplicon sequence variation. Each of these portions of information conveyed by SSR marker data was analysed as a separate source of variation. Results were compared across data sets to assess their evolutionary information content. Indels were coded as SNP mutations to represent them as single mutational events.For each data set (A) or haplotypes (h), Nei’s genetic diversity (eH), number of SNPs and indels) were recorded for each source of variation. Linkage disequilibrium (LD) was computed for FR sequence variation.Levels of polymorphism  for all data sets, at the level of individual alleles; (b) for Citrus loci CCT01 and GT03, which displayed extremely complex repeat sequences; see At the individual-allele level, matrices of pairwise genetic distances were computed between individuals and between alleles based on each source of variation. For amplicon length, distance is represented by difference in length; for SSR length, distance is the difference in number of repeat units; for whole amplicon sequence, distance is the total number of differences, including SNPs, indels and number of repeat units; for flanking region sequences, distance is the total number of SNPs and indels. The correlation between matrices was tested by a Mantel test. LD between all pairs of polymorphic sites in FR sequences was tested with a Markov chain Monte Carlo (MCMC) procedure of 10,000 steps and a burn-in phase of 1,000 steps . Pairwise genetic distances for amplicon and FR sequence variation were computed based on the number of mutational steps between sequences. Pairwise genetic distances for SSR variation and amplicon size variation data were computed as Euclidian distances  to account for their faster mutation rate. Weight choice was based on the observation that SSRs have, in our Jacaranda sample, approximately ten times more alleles than each SNP (see Results section). Considering that rare alleles might be missing from our sample, we estimated a ratio of 1∶20 in allele richness between SNPs and SSRs and used this ratio to establish the relative weight. A hierarchical analysis of molecular variance (AMOVA) was performed to partition genetic variance into within- and among-population components. The significance of variance components was tested by 1,000 permutations ST, RST, and NST matrices were used to construct population-level consensus UPGMA (Unweighted Pair Group Method with Arithmetic Mean) cladograms by averaging genetic distance information of three loci with SplitsTree4 v4.6 At the population level, for Citrus, Jacaranda, and Quercus data sets, respectively. Three, three and one SSRs were genotyped and sequenced for the Citrus, Jacaranda and Quercus data sets, respectively. The lengths of the sequenced fragments varied between 123 and 255 bp , sequence data produced from three divergent tree genera were evaluated. Forty-six, 101, and 47 samples constituted the d 255 bp . WheneveLevels of polymorphism  for each amplicon and each source of variation are displayed in Jacaranda and Quercus data set, An example of the complexity of the polymorphisms observed is shown for markers Jc3H10 and QrZAG30  showed significant LD. For the three SSR loci, LD was very irregular and there was no relationship between LD and sequence distance . For the Jacaranda data set, ten pairs out of 27 (37%) showed significant LD. For marker Jc3F4, the SNP at position 81 was in disequilibrium with two indels and one SNP, all located upstream of the SSR. LD was strongest for Jc3H10, where all three pairs were significant. The relationship between LD and sequence distance was significant . Tests of correlation between LD and distance in bp could not be performed for Jc3A10 and Jc3H10 because only three nucleotide sites were polymorphic . For the Quercus data set (marker QrZAG30), 146 pairs of SNPs and indels out of 325 (44.9%) showed significant LD. In particular, 13 or 14 contiguous SNPs and indels located downstream of the SSR at positions 84–107 formed a disequilibrium block, itself in weaker disequilibrium with upstream polymorphic sites at positions 23, 45 and 46 and another SNP downstream the SSR (position 60).LD was tested for all pairs of SNP and indel sites within the FR sequence variation of each marker . For theWe tested whether different sources of variation conveyed the same information on relatedness among alleles. To do so, genetic distances were computed for each pair of individuals with each source of variation  within each marker, and the correlation between genetic distance matrices was tested by a Mantel test. Among the 36 pairs of matrices, 27 (75%) showed a significant correlation . GeneticJacaranda data set, we tested population genetic differentiation for each source of variation by computing FST, RST and NST as appropriate  and SSR variation the smallest (0.05). RST values were very small (<0.02) and non-significant when computed on both amplicon size variation and SSR variation. On the contrary, NST values were large and significant, with values of 0.50 for FR sequence variation and 0.36 for amplicon sequence variation. In the phylogenetic trees obtained from each combination of source of variation/genetic distance measures, FST and NST provided the same topology for all cases they were applied to, indicating a more or less close link between pairs of populations Tapajos/Saint-Laurent and Counami/Paracou (ST grouped Paracou with Tapajos and Counami with Saint-Laurent (for amplicon size variation) or separated the three Guiana shield populations from Tapajos . A total of seven (dinucleotide and trinucleotide) SSR markers were targeted, including compound and imperfect markers. More genetic diversity was found for amplicon sequence variation than for amplicon size variation, suggesting that much of data polymorphism is neglected when SSRs are described through amplicon size alone. On average, 7.7 SNPs and 3.4 indels were detected in the flanking sequences of each marker. Genetic diversity (eH) ranged between 0.306 and 0.922 for the FRs. LD among SNPs and indels in the FR was generally significant. These results demonstrate that SSR alleles are very often riddled with abundant non-SSR indels, plus vast amounts of SNPs both within and near the repeat itself. Similar genetic variation was previously observed at intra- or inter-specific levels SSR polymorphisms are widely used to infer population history and biogeographic patterns. These inferences rely on assumptions about SSR mutational models, and departures from such models are likely to bias divergence and diversity estimates. SSR alleles have been demonstrated to contain other sources of polymorphism in addition to variation in repeat number. In this study we have characterised the contribution of molecular variation occurring outside SSR repeats to SSR marker variability. Whole allele sequencing from SSR markers was analyzed from three tree genera  a strictly geographical clustering (the three Guiana shield populations cluster and are separated from the Amazonian population), and (b) a pattern derived from independent results on chloroplast DNA divergence . Our results support the latter hypothesis, as proven by the convergent topology of trees obtained with FST and NST independently of the source of variation. The purely geographical hypothesis, on the other hand, is only supported by SSR variation in combination with RST . RST estimates have larger variance than FST and NST, particularly when based on small numbers of loci, and RST values obtained here may be unreliable. Nevertheless, the pattern of RST differentiation closely follows geographical distance. This may indicate that repeat number variation has arisen locally from small numbers of founding alleles and reflects recent population divergence, while variation in flanking regions may follow larger phylogeographic patterns and reflect deeper divergence. If RST estimated can be trusted, all sources of variation converge with independent, chloroplast-based results, except SSR variation itself. Therefore, information derived from the “true” nature of SSR data (SSR variation) is actually not representative of the information conveyed by alleles as a whole. This finding strongly hints that other sources of variability, more correctly described by mutation models that apply to DNA sequences, actually provide the bulk of information carried by SSRs. In other words, phylogeographic information carried by amplicon length is not provided by the SSR (which amplicon length is intended to represent) but by variation in flanking regions. Note that this contrasts with analyses carried out at the individual level (see above), where information on genetic distance match when measured as amplicon size and as SSR size. This issue raises the question of the application of the SMM, or other step-based mutational models, to SSRs, as already discussed by Colson and Goldstein et al.Jacaranda SSR markers and by the Citrus SSR marker GT03 The consequences – and usefulness – of hidden sequence variation are shown when we compare how sources of variation perform in detecting population divergence. This analysis was performed in the Jacaranda data set (but larger numbers of markers may reduce estimation variance and mitigate this problem). Taking sequence variation into account actually adds a significant piece of information to phylogenetic or phylogeographic reconstruction. Population structuring emerges more clearly on the basis of sequence data, and the combination of sequences and SSR variation provides higher resolution. Consequently, genetic distance measures assuming the SMM or related models should be restricted to perfect SSRs with invariable FRs. On the other hand, measures such as FST (for amplicon size data) and NST (for amplicon sequences) seem to be the tool of choice for the analysis of the average, not-so-ideal SSR markers that a population geneticist meets in his or her everyday work.Given the complexity of amplicon sequences described here, one may wonder how to properly exploit SSR data. The weight of polymorphisms other than variation in repeat number cannot and should not be overlooked: identity in allele size does not necessarily indicate identity in sequence content or the number of repeats within same sized alleles. When considering individual alleles, amplicon size correlates well with SSR repeat number. Nevertheless, even if repeat number could be obtained directly, it may provide only weak and inconsistent signals of population genetic differentiation, as shown by the Figure S1Complete alignments of DNA fragments for all SSR markers studied. Marker names are shown above each alignment.(PDF)Click here for additional data file.Figure S2Z-statistics andsummary of Mantel tests for the correlations of pairs of genetic distance matrices within species. Colouring (shading) indicates the level of significance of the test: green (light grey*), P>0.05; orange (grey*), 0.05<P>0.001; red (dark grey*), P<0.001. Identity of DNA fragment sections is reported: simple sequence repeat (SSR) variation; amplicon size variation, flanking region (FR) sequence variation, and amplicon sequence variation. Marker names are displayed above each panel and data sets refer to Citrus (C), Jacaranda (J) and Quercus (Q). Sequences with single nucleotide polymorphisms (SNPs) within the repeat were excluded from subsequent analyses because of the complexity to describe the mutation model of SSR repeats bracketing SNPs.(PDF)Click here for additional data file.Table S1List and details of the sampled sites for the Jacaranda (J) data set.(DOC)Click here for additional data file.Table S2Description, amplification conditions and polymorphism of the nuclear simple sequence repeats (SSRs) analysed in Jacaranda copaia.(DOC)Click here for additional data file.Table S3List and details of alleles for the three data sets. Amplicon sizes, frequencies and number of associated haplotypes are indicated for each allele.(DOC)Click here for additional data file.