For approximately four decades, scientists have known of the existence of several undescribed species of Siren in the southeastern United States Coastal Plain. One of these species, S. reticulata, was recently described, but a small, seepage-dwelling species has remained undescribed until now. To resolve outstanding questions concerning the phylogeny of Siren, we collected sequence and morphometric data from specimens across the range of Siren. We found S. lacertina and S. reticulata to represent strongly supported monophyletic groups, with S. reticulata having a sister relationship to all other Siren. Additionally, we found five distinct mtDNA lineages within what has been recognized as S. intermedia. Siren lacertina and type-locality S. intermedia (lineage A) are sister mtDNA lineages, whereas S. intermedia lineages B and C show a high level of mitogenomic divergence from type-locality S. intermedia. Analyses of two scnDNA loci revealed that S. lacertina is monophyletic but nested with low positional support in a clade including the three S. intermedia mtDNA lineages. Further study is needed to determine whether S. intermedia lineages A, B, and C represent distinct species or incompletely sorted lineages. We restrict the range of S. intermedia to the region from the Escambia and Perdido river drainages of Florida and Alabama eastward through Virginia (the combined ranges of lineages A, B, and C). We also elevate S. i. nettingi (lineage E) to species status and include the larger S. i. texana form in that taxon, generating a species that occurs from the Mobile Bay drainages westward through the Mississippi Basin and southwest into northeastern Mexico. Lastly, we describe a new miniature species, S. sphagnicola, that ranges from the Florida Parishes of Louisiana eastward to the westernmost tributary creeks of Choctawhatchee Bay in the western Florida panhandle.

[This corrects the article DOI: 10.1371/journal.pone.0044821.].

Ovipleistophora (Microsporidia) is a globally distributed genus of obligate parasites that infect fish, Crustacea, and trematodes. We report on two additional crayfish hosts, Procambarus pictus (Simms Creek, Florida) and Procambarus fallax (Santa Fe River, Florida), that exhibited signs of high-intensity microsporidian infection in the musculature. Sequence data (SSU) for the isolates were 99.79% and 99.97% similar to Ovipleistophora diplostomuri parasitizing Procambarus bivittatus. Additional screening of regional fish species (Lepomis macrochirus) revealed additional microsporidian diversity (Potaspora) but did not include Ovipleistophora. Detecting Clade V isolates in multiple host groups provides further evidence for trophic transmission among Clade V microsporidia.

The genus Procambarus represents a diverse genus of freshwater crayfish that includes epigean species, stygobitic species, and at least one parthenogenic species. Despite its evolutionary, ecological, and economic importance, most genomic and transcriptomic resources for this genus are limited to a couple of model species. We sequenced the transcriptome of a non-model species, P. erythrops, a geographically restricted stygobitic species from Florida. RNA isolated from gill, muscle and eye tissue was pooled to create a de novo transcriptome assembly using Single Molecule Real-Time sequencing (PacBio), resulting in 19,442 full-length isoforms. The assembly has been deposited in the NCBI (BioProject PRJNA657230). These data will make an important contribution to the comparative study of transcriptome evolution in crayfish and crustaceans.

The Suwannee alligator snapping turtle (Macrochelys suwanniensis) was first described in 2014. The species is thought to occur in blackwater and spring-fed streams of sufficient size throughout the Suwannee River drainage, but we have limited detailed information regarding its range. To clarify the distribution of M. suwanniensis, we compiled 111 museum, 16 literature, and 40 other credible records and trapped streams throughout the Suwannee drainage in Georgia and Florida, plus 8 streams in the Big Bend region of Florida (total of 1893 trap nights). We documented the first records from the Willacoochee River and Okapilco, Piscola, Warrior, Jones, and Toms creeks in Georgia and from Rocky and Olustee creeks in Florida. Relative abundance based on catch per unit effort (CPUE) varied among streams (0.00-0.50) and sections of the same stream. Macrochelys suwanniensis is apparently scarce in the Okefenokee Swamp and in the Suwannee River upstream of White Springs, Florida (none trapped), but it occurs in small blackwater tributaries in this section of the river in both states. In the Suwannee River between White Springs and the estuary, we had a mean CPUE of 0.25, and the highest trapping success in Florida was in downstream reaches of the Suwannee River and in its major tributaries, the Santa Fe and New rivers. The species is widely distributed in Georgia, but relative abundance in most streams appears lower than in Florida. In Georgia, we had the highest trapping success in the Alapaha drainage and in sections of the Little River and Okapilco Creek in the Withlacoochee drainage. In Florida, we failed to trap Macrochelys in the purported distribution gap between the Suwannee and Ochlockonee drainages and in the Wacasassa River to the south, indicating that this species is restricted to the Suwannee drainage.

Loss of wetlands throughout the southeastern United States threatens the persistence of the region's highly diverse freshwater fauna. Losses are especially concerning for rare species that maintain small or fragmented ranges, zoogeographies that characterize many of the region's numerous freshwater endemics. We assayed nuclear and mitochondria' DNA sequence data from the Pine Barrens Treefrog (Hyla andersonii), a rare species distributed across the Atlantic and Gulf coastal plains. We hypothesized that the species' evolutionary history has been associated with changes in wetlands during Quaternary interglacials and that the contemporaneous extent of wetlands is positively correlated with population genetic diversity. Genetic variation was highest in North Carolina and South Carolina and lowest in New jersey and Florida. Mean times to common ancestry ranged from 132,486 to 1,290,605 yr before present, and effective sizes ranged from 4,241 individuals in New Jersey to 403,718 individuals in North Carolina. Population migration rates were generally very low (<0.01), although higher rates were found between North Carolina and South Carolina. Total area of wetlands varied from 2,482 km(2) in South Carolina to 7,384 km(2) in North Carolina and has declined between 2001 and 2016. Genetic diversity was positively, although nonsignificantly, correlated to total amount of wetland habitat. Pine Barrens Treefrog is comprised of four relictual populations associated with ecological changes driven by climatic progressions of Quaternary interglacials, and collectively these populations conform to an abundant center model of evolution. All populations are conservatively designated management units, although evolutionarily significant unit status cannot be discounted.

Two species of purseweb spiders. Sphodros abboti (Blue Purseweb Spider) and S. rufipes (Red-legged Purseweb Spider), are known to occur in northern Florida and southern Georgia, and a third, S. atlanticus (Atlantic Purseweb Spider), has been recorded from northeastern Georgia. Their distributions have not been well defined, and records are sparse for S. rufipes and S. atlanticus. We conducted visual surveys for the distinctive tubular webs of these spiders in a variety of forest types. We report 141 new locality records for S. abboti, including 19 new county records in Florida and 5 in Georgia . , 80 new locality records for S. rufipes, including 13 new county records in Florida, 34 in Georgia, and 1 in Alabama; and 3 new locality records for S. atlanticus, including 2 new county records for Georgia. We provide additional information on reproductive biology and habitat use.

Abstract We conducted roadside call surveys at 111 historical Hyla andersonii (Pine Barrens Treefrog) sites in 2013–2016. We detected the species at 49 sites (44%), which is consistent with surveys conducted in the early 1980s. We detected the species throughout its historical range except Holmes County, which only has 4 historical sites. We also identified 33 new sites on public lands and 4 new sites on private lands. We used multi-season occupancy models to determine the influence of site and survey covariates on species occupancy and detection rates in Blackwater River State Forest. Species detection was positively associated with air temperature and partial cloud-cover and negatively associated with clear skies. Pine Barrens Treefrog occupancy rates were lower at survey sites surrounded by hardwood-dominated wetlands, suggesting that hardwood encroachment has negatively impacted this species on some areas of Blackwater River State Forest. The best model had an estimated detection probability of 0.43 (95% CI = 0.29–0.47) and an estimated occupancy of 0.63 (95% CI = 0.43–0.79). We recommend that future call surveys include a minimum of 5 visits to each survey site before inferring species absence. Our surveys suggest that the status and distribution of the species remain stable despite some habitat degradation.

Crayfish are a vital ecological asset in their native range but can be highly damaging as invasive species. Knowledge of their diseases, including high levels of research on Aphanomyces astaci (crayfish plague), show that disease plays a vital role during crayfish invasions. Microsporidian diseases in crayfish are less studied but are thought to have important links to crayfish health and invasion dynamics. In this study we provide a systematic description of a novel microsporidian parasite from the Floridian crayfish, Procambarus paeninsulanus, with additional genetic identification from related Microsporidia from Procambarus fallax, Cambarellus shufeldtii and Cambarellus blacki. This novel microsporidium from P. paeninsulanus is described in a new genus, Cambaraspora, and species, Cambaraspora floridanus, and represents a novel member of the Clade V Microsporidia within the Glugeidae. The parasite develops in the muscle tissue of P. paeninsulanus, within a sporophorous vesicle, and produces a spore with 19-21 turns of the polar filament measuring 6.136 ± 0.84 µm in length and 2.12 ± 0.23 µm in width. The muscle-infecting nature of the parasite suggests that it is horizontally transmitted. Genetic data for the 18S of the parasite from all hosts confirms its assignment to Clade V and reveal it to be a relative of multiple fish-infecting parasites. It shows closest genetic relationship to Glugea plecoglossi, but branches alongside multiple microsporidia from fish, crustaceans and eDNA isolates. The information presented here suggests that this novel parasite may have the potential to infect piscine hosts and is a likely mortality driver in the P. paeninsulanus population. Its potential as a control agent or wildlife disease invasion threat is explored, as well as the placement of this novel microsporidium within the Glugeidae.

A new species of Calamaria Boie, 1827 is described based on a single specimen collected in evergreen forest at 1240 m elevation of Ta Dung Nature Reserve in Dak Nong Province, Central Vietnam. The new species is characterized by (1) rostral wider than high; (2) paraparietal surrounded by six shields and scales; (3) eye diameter larger than eye-mouth distance; (4) preocular present; (5) supralabials 5/4, 3-4/2-3 entering orbit; (6) maxillary teeth nine, modified; (7) infralabials 5/4, first three touching anterior chin shields; (8) mental touching tip of right anterior chin shield; (9) ventrals 1 + 174; subcaudal scales 18/17, divided; (10) precloacal plate single; (11) tail relatively short (6.2% of the total length), nearly as thick as body, slightly tapering, and ending in obtuse point; (12) dorsal scales reducing to six rows at position above 4th subcaudal, and to four rows above 13th subcaudal on tail; (13) dorsum dark with irregular yellow blotches; and (14) ventral side dark with few yellow blotches and bands. This is the sixth new Calamaria species described from Vietnam in the past thirteen years and the tenth species of Calamaria recorded from this country.

Ovipleistophora diplostomuri (Microsporidia) is an obligate parasite of fish and trematodes in the US. In April 2019, an individual crayfish, Procambarus bivittatus (Escambia River, Florida), with a high-intensity microsporidian infection was delivered to the Emerging Pathogens Institute. Histological analysis determined that infection was restricted to the muscle tissue. Molecular diagnostics (PCR) provided 952 bp of the parasite SSU (18S) sequence. The isolate was 99.16% similar to O. diplostomuri identified from blue gill and their trematode parasites in Washington, USA. This discovery increases our understanding of Microsporidia within aquatic trophic networks, supporting the theory that the Ovipleistophora share complex relationships with vertebrates, invertebrates and helminth parasites.

Abstract Florida scrub is a xeric ecosystem associated with the peninsula's sand ridges, whose intermittent Pliocene–Pleistocene isolation is considered key to scrub endemism. One scrub origin hypothesis posits endemics were sourced by the Pliocene dispersal of arid‐adapted taxa from southwestern North America; a second invokes Pleistocene migration within eastern North America. Only one study to date has explicitly tested these competing hypotheses, supporting an eastern origin for certain scrub angiosperms. For further perspective, we conducted a genetic analysis of an endemic arthropod, the Florida sand cockroach (Arenivaga floridensis), with two aims: (1) to reconstruct the peninsular colonization and residence history of A. floridensis and (2) determine whether its biogeographic profile favors either origin hypothesis. We sequenced the cox2 mitochondrial gene for 237 specimens (65 populations) as well as additional loci (cox1, nuclear H3) for a subset of Florida roaches and congeners. Using Network and Bayesian inference methods, we identified three major lineages whose genetic differentiation and phylogeographical structure correspond with late Pliocene peninsula insularization, indicating Arenivaga was present and broadly distributed in Florida at that time. Stem and crown divergence estimates (6.36 Ma; 2.78 Ma) between A. floridensis and western sister taxa span a period of extensive dispersal by western biota along an arid Gulf Coast corridor. These phylogeographical and phylogenetic results yield a biogeographic profile consistent with the western origin hypothesis. Moreover, age estimates for the roach's peninsular residence complement those of several other endemics, favoring a Pliocene (or earlier) inception of the scrub ecosystem. We argue that eastern versus western hypotheses are not mutually exclusive; rather, a composite history of colonization involving disparate biotas better explains the diverse endemism of Florida scrub.

Necturus beyeri (Caudata: Proteidae), as conceived by some, contains paedomorphic salamanders distributed from the Ochlockonee drainage of Florida to the Angelina drainage of Texas. Because these salamanders differ in color pattern and karyotype across their geographic range, we performed a phylogeographic analysis that included representatives from all major drainages as well as of all congeners. The mitochondrially encoded ND2 gene was used to infer phylogenetic relationships using Bayesian inference. Morphometrics of head shape were analyzed and included as an independent data set. Our work suggests that Necturus comprises 11 lineages. A basal split within the genus separates an ancestor of two Atlantic Coastal Plain species (Necturus lewisi and Necturus punctatus) from the ancestor of nine distinct Gulf Coastal Plain lineages. One lineage is consistent with Necturus alabamensis, a species currently recognized in the Black Warrior drainage of Alabama. Two lineages comprise Necturus maculosus, as historically recognized, and six lineages comprise N. beyeri, as recognized by some, each of which occupies a unique drainage. Both of these species are demonstrated to be paraphyletic. Head morphometrics show the same patterns as the mtDNA. Overall, lineages within Necturus exhibit an east-to-west progression of appearance on the phylogenetic trees. This pattern corroborates biogeographic hypotheses based on previous karyological work. Within N. beyeri, this progression separates a pattern class of two eastern lineages lacking bold spotting and possessing relatively small mean body lengths from a pattern class of four western lineages possessing bold spotting and larger mean body sizes. Thus, the two eastern lineages of N. beyeri are similar in color pattern and body size to N. punctatus either through retention of the ancestral color pattern and size for the genus or through convergent selection in eastern streams of the Gulf Coastal Plain.

The Alligator Snapping Turtle, Macrochelys temminckii, is a large, aquatic turtle limited to river systems that drain into the Gulf of Mexico. Previous molecular analyses using both mitochondrial and nuclear DNA suggested that Macrochelys exhibits significant genetic variation across its range that includes three distinct genetic assemblages (western, central, and eastern = Suwannee). However, no taxonomic revision or morphological analyses have been conducted previously. In this study, we test previous hypotheses of distinct geographic assemblages by examining morphology, reanalyzing phylogeo-graphic genetic structure, and estimating divergence dating among lineages in a coalescent framework using Bayesian inference. We reviewed the fossil record and discuss phylogeographic and taxonomic implications of the existence of three distinct evolutionary lineages. We measured cranial (n= 145) and post-cranial (n= 104) material on field-captured individuals and museum specimens. We analyzed 420 base pairs (bp) of mitochondrial DNA sequence data for 158 Macrochelys. We examined fossil Macrochelys from ca. 15-16 million years ago (Ma) to the present to better assess historical distributions and evaluate named fossil taxa. The morphological and molecular data both indicate significant geographical variation and suggest three species-level breaks among genetic lineages that correspond to previously hypothesized genetic assemblages. The holotype of Macrochelys temminckii is from the western lineage. Therefore, we describe two new species as Macrochelys apalachicolae sp. nov. from the central lineage and Macrochelys suwanniensis sp. nov. from the eastern lineage (Suwannee River drainage). Our estimates of divergence times suggest that the most recent common ancestor (MRCA) of M. temminckii (western) and M. apalachicolae (central) existed 3.2-8.9 Ma during the late Miocene to late Pliocene, whereas M. temminckii-M. apalachicolae and M. suwanniensis last shared a MRCA 5.5-13.4 Ma during the mid-Miocene to early Pliocene. Examination of fossil material revealed that the fossil taxon M. floridana is actually a large Chelydra. Our taxonomic revision of Macrochelys has conservation and management implications in Florida, Georgia, and Alabama.

With the exception of the Malay Peninsula, onychophorans have only recently been reported from mainland Southeast Asia. Here we report the second record of an onychophoran from Vietnam. K EY WORDS Onychophora, Vietnam, Quang Ngai, Southeast Asia, Truong Son Mountains

Habitat loss, degradation, and fragmentation are thought to be the primary causes of declines in the distribution and abundance of the eastern indigo snake (Drymarchon couperi) throughout its range, although no long-term studies have verified these effects. We report dramatic declines in the relative abundance of this species in the gulf hammock of levy county, Florida, USA. radio-tracking of D. couperi in an 8,628-ha portion of the gulf hammock Wildlife management area from 1981 to 1983 documented catch-per-unit-effort and habitat conditions and use by snakes. using similar sampling methods (road cruising and visual encounter surveys), an intensive survey on the same study area from 2005-2009 indicated that the number of individual indigo snakes observed per field day and per field hour had declined by 97.6% and 98.9%, respectively. Potential indigo snake habitat did not become more fragmented, decrease in total amount, or substantially change in cover types in our study area or regionally over these three decades. circumstantial evidence suggests that cumulative, unsustainable mortality from vehicular traffic, intentional killing, and perhaps intensive forestry operations contributed significantly to the population crash. Predation of nests by red imported fire ants (Solenopsis invicta) also may be important, but environmental pollution, disease, and climate change seem to be insignificant or discountable factors at this site. similar environmental conditions may be present over much of the remaining range of this threatened species.

Records from a colubrid host are reported for Hepatozoon horridus, described originally from a viperid snake. Hepatozoon horridus in Pantherophis guttatus (Colubridae) has gamonts 14-18.0 by 4.0-5.5 microm, with length by width (LW) 60-99 microm2, and L/W ratio 2.5-3.9. Spherical to elongate, usually ovoid oocysts with L/W ratio 1.0-3.7 contain 16-160 spherical to usually ovoid sporocysts 15-31 by 14-26 microm, with L/W ratio 1.0-1.4, that contain 5-24 sporozoites. Two additional Hepatozoon species are described from ratsnakes in north Florida. Hepatozoon quadrivittata n. sp. from Pantherophis obsoletus quadrivittatus has gamonts 12-17 by 4-6 microm, LW 56-102 microm2, and L/W ratio 2.6-3.8. Nearly spherical oocysts with L/W 1.0-1.1 contain 5-227 spherical to slightly ovoid sporocysts 20-48 by 19-45 microm, with L/W ratio 1.0-1.4, that contain 13-48 sporozoites. Hepatozoon spiloides n. sp. from Pantherophis obsoletus spiloides forms gamonts 12-15 by 4-5 microm with LW 48-75 microm2 and L/W ratio 2.6-3.5. Occasionally rounded but usually elongate oocysts, with L/W ratio 1.0-2.7, contain 5-21 spherical to elongate sporocysts 28-43 by 18-35 microm, L/W ratio 2.5-3.9. In the distinctive Hepatozoon sp. present in Pantherophis obsoletus spiloides, gamonts are 13-17 by 5-10 microm, with LW 75-140 microm2 and L/W ratio 1.4-3.0. Infected erythrocytes are always distorted and enlarged on average 2.5 times the size of uninfected cells, with nuclei enlarged by one-third and broadly elongated. Gamonts often stained deep blue, and cytoplasm of erythrocytes infected with mature gamonts was always dehemoglobinized. Sporogony could not be obtained in three feedings by hundreds of Aedes aegypti, which usually died within the first 24-48 hr.

When a widespread species is sympatric with a rare, geographically restricted conspecific, recurrent gene flow can pose the risk of extinction for the latter. This can occur via genetic swamping, where the occurrence of hybrids eventually replaces the numerically less abundant species. We took a molecular genetic approach to quantify the occurrence and degree of contemporary hybridization between two species of frog co-occurring in a small geographic area in northwest Florida, USA. The Florida bog frog Lithobates okaloosae is a small ranid limited in distribution to a few acidic seepage and steephead streams that feed the Yellow, Shoal and East Bay river drainages in Walton, Okaloosa and Santa Rosa counties, Florida. The bronze frog Lithobates clamitans is a widespread (Eastern North America) congener. Data from nine microsatellite loci and 350 frogs were analyzed using Bayesian clustering (structure) and a Bayesian hybrid classification method implemented in newhybrids. Power to detect hybrids with the dataset was assessed through simulations. Both methods detected hybrids in similar proportions (5–10%), including congruent confirmation and rejection of samples previously classified as putative hybrids based on phenotypic characteristics. However, the nine loci lacked sufficient power to differentiate among F1 and backcrossed individuals, leaving open the question of the extent of genetic introgression. Longitudinal genetic monitoring is recommended to evaluate whether the observed level of hybridization represents a long-term threat to the distinction of one of North America's most geographically restricted frogs.

Cambarus (Lacunicambarus) miltus (Rusty Gravedigger Crayfish) is a primary burrowing crayfish known from a limited portion of the Gulf Coastal region of the United States. The lack of form I males in collections has in the past prevented species-level identifications and hampered conservation reviews. We conducted an intensive status survey for C. miltus during 2007 and 2008. Our results suggest that the species is much more widespread than previously known and that conservation attention is unwarranted. Preferred habitat for the species is ephemerally flooded and thinly wooded floodplains of small streams and swamps.