Unraveling Siren (Caudata: Sirenidae) systematics and description of a small, seepage specialist.

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.

A biogeographical profile of the sand cockroach Arenivaga floridensis and its bearing on origin hypotheses for Florida scrub biota.

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.

Taxonomic assessment of Alligator Snapping Turtles (Chelydridae: Macrochelys), with the description of two new species from the southeastern United States.

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 phylogeographic 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.

Four additional hepatozoon species (Apicomplexa: Hepatozoidae) from north Florida ratsnakes, genus Pantherophis.

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.

Evolution of gigantism in amphiumid salamanders.

The Amphiumidae contains three species of elongate, permanently aquatic salamanders with four diminutive limbs that append one, two, or three toes. Two of the species, Amphiuma means and A. tridactylum, are among the largest salamanders in the world, reaching lengths of more than one meter, whereas the third species (A. pholeter), extinct amphiumids, and closely related salamander families are relatively small. Amphiuma means and A. tridactylum are widespread species and live in a wide range of lowland aquatic habitats on the Coastal Plain of the southeastern United States, whereas A. pholeter is restricted to very specialized organic muck habitats and is syntopic with A. means. Here we present analyses of sequences of mitochondrial and nuclear loci from across the distribution of the three taxa to assess lineage diversity, relationships, and relative timing of divergence in amphiumid salamanders. In addition we analyze the evolution of gigantism in the clade. Our analyses indicate three lineages that have diverged since the late Miocene, that correspond to the three currently recognized species, but the two gigantic species are not each other's closest relatives. Given that the most closely related salamander families and fossil amphiumids from the Upper Cretaceous and Paleocene are relatively small, our results suggest at least two extreme changes in body size within the Amphuimidae. Gigantic body size either evolved once as the ancestral condition of modern amphiumas, with a subsequent strong size reduction in A. pholeter, or gigantism independently evolved twice in the modern species, A. means and A. tridactylum. These patterns are concordant with differences in habitat breadth and range size among lineages, and have implications for reproductive isolation and diversification of amphiumid salamanders.

A new Haemogregarina species of the alligator snapping turtle, Macrochelys temminckii (Testudines: Chelydridae), in Georgia and Florida that produces macromeronts in circulating erythrocytes.

Haemogregarina macrochelysi n. sp. (Apicomplexa: Haemogregarinidae) of the alligator snapping turtle, Macrochelys temminckii, is characterized by slender, recurved gamonts 29-35 x 3-4.5 microm, in which the anterior limb comprises 48-54% of the total length. The gamont nucleus, 5-7.5 x 2-5 microm, is situated at approximately midbody of the gamont. Meronts typical of Haemogregarina occupying erythrocytes have 3-8 small, compact nuclei and are 13-17 x 4.5-9 microm. Erythrocytic meronts that contain larger, nearly square or rectangular nuclei become rounded, and then undergo 7 or more nuclear divisions, which produce very large, usually ovoid to rounded meronts that may contain up to 150 nuclei or more within the thinly stretched host erythrocyte membrane. In tissues of the Placobdella spp. leech vectors, merogony occurs directly from sporozoites, forming merozoites that presumably are infective for the turtle host.

Hepatozoon species of the timber rattlesnake in northern Florida: description of a new species, evidence of salivary gland oocysts, and a natural cross-familial transmission of an Hepatozoon species.

Two species of Hepatozoon, i.e., H. sauritus and H. horridus n. sp., were present in 1 of 8 timber rattlesnakes, Crotalus horridus. The narrow gamonts of H. sauritus are 15.0-19.0 x 3.5-5.0 microm, with LW 58-86 microm2 and L/W 3.2-4.7, with a narrow, rounded anterior end. The spherical to slightly ovoid oocysts produce ovoid to elongate sporocysts, 21-43 x 12-24 microm, L/W 1.20-2.7, containing on average 22.1 (10-34) sporozoites. This is the first report of a natural cross-familial transfer of a Hepatozoon species. Gamonts of H. horridus n. sp. are 13.0-17.0 x 4.0-6.0 microm, with LW 63-102 microm2 and L/W 2.6-4.0, and have broadly rounded ends. The gamont cytoplasm is vacuolated. The spherical to ovoid oocysts form spherical to elongate sporocysts 14-45 x 11-25 microm, L/W 1.0-2.3, producing an average of 13.0 (8-21) sporozoites. The salivary gland in 1 of 5 mosquitoes dissected contained 1 mature oocyst.

Peptidomic analysis of skin secretions from Rana heckscheri and Rana okaloosae provides insight into phylogenetic relationships among frogs of the Aquarana species group.

The members of the Aquarana (or Rana catesbeiana species group) form a monophyletic group comprising seven species: R. catesbeiana, Rana clamitans, Rana grylio, Rana virgatipes, Rana septentrionalis, Rana heckscheri and Rana okaloosae. Previous work has led to structural characterization of the antimicrobial peptides present in electrically-stimulated skin secretions from the first five species listed and this study presents the primary structures of orthologs from the river frog R. heckscheri and the Florida bog frog R. okaloosae. Peptidomic analysis of R. heckscheri and R. okaloosae skin secretions led to the identification of peptides with antimicrobial activity belonging to the ranalexin, ranatuerin-2, and temporin families. In addition, a peptide (GFLDIIKDTGKDFAVKILNNLKCKLAGGCPR) was isolated from R. okaloosae whose primary structure identified it as a member of the palustrin-2 family. Consistent with previous data based upon morphological analysis and comparisons of the nucleotide sequences of mitochondrial and ribosomal genes, cladistic analysis based upon a comparison of the amino acid sequences of antimicrobial peptides indicates a sister-group relationship between R. heckscheri and R. grylio and a close, but less well defined, phylogenetic relationship between R. okaloosae and R. clamitans.

Phylogeography of the salamander genus Pseudobranchus in the southeastern United States.

This study provides an extensive set of mitochondrial cytochrome b sequences for the salamander genus Pseudobranchus of the southeastern United States. These sequences were analysed by multiple phylogenetic methods that support a single set of major phylogeographic divisions for its two corroborated species (P. axanthus and P. striatus). These phylogeographic divisions overlap with the geographic breakpoints of other freshwater and terrestrial taxa in this region. Collectively, these overlapping patterns highlight the Central Highland and Tifton/Vidalia uplands as a significant barrier to Atlantic vs. Gulf coast groups, while reconfirming the phylogeographic significance of the Altamaha and Apalachicola river drainages. Despite their distinct phylogeographic split, P. striatus from west and east of these uplands are not currently recognizable as separate species according to the concordance principles for species definition.

Two additional Hepatozoon species (Apicomplexa: Hepatozoidae) from the southern black racer, Coluber constrictor priapus (Serpentes: Colubridae), in northern Florida.

Hepatozoon priapus n. sp. from Coluber constrictor priapus has robust gamonts with broadly rounded ends, 18.0 x 4.2 microm (17.0-20.0 x 3.5-6.0), with LW 76.4 microm2 (59-105) and L/W 4.31 (2.9-5.4). The nucleus is always present in second quarter of gamont, seldom extend into first quarter but often into third quarter, 6.0 x 3.0 (5.0-7.0 x 2.5-4.0), with LW 17.9 (13.7-21.0). Erythrocyte cytoplasm is always thin, appearing dehemoglobinized, with infected cells always distorted. Infected erythrocytes are much longer and wider than uninfected cells, with longer nuclei. Oocysts are spherical to ovoid, 92.5 x 86.0 (55-123 x 47-115) and L/W 1.08 (1.0-1.3), contain 14.0 (6-31) sporocysts. Sporocysts, which are also spherical to ovoid, 26.3 x 23.3 (19-50 x 16-38), LW 641.2 (320-1,500) and L/W 1.13 (1.0-2.2), contain 12.6 (5-18) sporozoites. Hepatozoon confusus n. sp., also from C. constrictor priapus, has slender gamonts with rounded ends, 15.6 x 4.1 (14.0-17.0 x 3.5-5.0), with LW 64.3 (52-80) and L/W 3.82 (2.8-4.4). The nucleus is always present in second quarter of gamont, commonly extending into first and third quarters, 5.0 x 2.7 (2.5-4.4 x 4.0-6.0), with LW 13.5 (11.0-16.5). Erythrocyte cytoplasm is sometimes thin, appearing partially dehemoglobinized, with infected cells usually distorted. Infected erythrocytes are longer than uninfected cells but similar in width, with erythrocyte nuclei longer. Oocysts are spherical to ovoid, 115.5 x 108.9 (52-278 x 50-278), with L/W 1.06 (1.0-1.2), and contain 25.0 (7-111) sporocysts. Sporocysts are spherical to ovoid, 27.6 x 25.2 (21-38 x 20-33), LW 701.3 (420-1,125) and L/W 1.09 (1.0-1.4), containing 20.2 (12-32) sporozoites.