Climatic and topographic changes since the Miocene influenced the diversification and biogeography of the tent tortoise (Psammobates tentorius) species complex in Southern Africa.

BACKGROUND: Climatic and topographic changes function as key drivers in shaping genetic structure and cladogenic radiation in many organisms. Southern Africa has an exceptionally diverse tortoise fauna, harbouring one-third of the world's tortoise genera. The distribution of Psammobates tentorius (Kuhl, 1820) covers two of the 25 biodiversity hotspots in the world, the Succulent Karoo and Cape Floristic Region. The highly diverged P. tentorius represents an excellent model species for exploring biogeographic and radiation patterns of reptiles in Southern Africa. RESULTS: We investigated genetic structure and radiation patterns against temporal and spatial dimensions since the Miocene in the Psammobates tentorius species complex, using multiple types of DNA markers and niche modelling analyses. Cladogenesis in P. tentorius started in the late Miocene (11.63-5.33 Ma) when populations dispersed from north to south to form two geographically isolated groups. The northern group diverged into a clade north of the Orange River (OR), followed by the splitting of the group south of the OR into a western and an interior clade. The latter divergence corresponded to the intensification of the cold Benguela current, which caused western aridification and rainfall seasonality. In the south, tectonic uplift and subsequent exhumation, together with climatic fluctuations seemed responsible for radiations among the four southern clades since the late Miocene. We found that each clade occurred in a habitat shaped by different climatic parameters, and that the niches differed substantially among the clades of the northern group but were similar among clades of the southern group. CONCLUSION: Climatic shifts, and biome and geographic changes were possibly the three major driving forces shaping cladogenesis and genetic structure in Southern African tortoise species. Our results revealed that the cladogenesis of the P. tentorius species complex was probably shaped by environmental cooling, biome shifts and topographic uplift in Southern Africa since the late Miocene. The Last Glacial Maximum (LGM) may have impacted the distribution of P. tentorius substantially. We found the taxonomic diversify of the P. tentorius species complex to be highest in the Greater Cape Floristic Region. All seven clades discovered warrant conservation attention, particularly Ptt-B-Ptr, Ptt-A and Pv-A.

Turtles and Tortoises Are in Trouble.

Turtles and tortoises (chelonians) have been integral components of global ecosystems for about 220 million years and have played important roles in human culture for at least 400,000 years. The chelonian shell is a remarkable evolutionary adaptation, facilitating success in terrestrial, freshwater and marine ecosystems. Today, more than half of the 360 living species and 482 total taxa (species and subspecies combined) are threatened with extinction. This places chelonians among the groups with the highest extinction risk of any sizeable vertebrate group. Turtle populations are declining rapidly due to habitat loss, consumption by humans for food and traditional medicines and collection for the international pet trade. Many taxa could become extinct in this century. Here, we examine survival threats to turtles and tortoises and discuss the interventions that will be needed to prevent widespread extinction in this group in coming decades.

Ancient mitogenomics clarifies radiation of extinct Mascarene giant tortoises (Cylindraspis spp.).

The five extinct giant tortoises of the genus Cylindraspis belong to the most iconic species of the enigmatic fauna of the Mascarene Islands that went largely extinct after the discovery of the islands. To resolve the phylogeny and biogeography of Cylindraspis, we analysed a data set of 45 mitogenomes that includes all lineages of extant tortoises and eight near-complete sequences of all Mascarene species extracted from historic and subfossil material. Cylindraspis is an ancient lineage that diverged as early as the late Eocene. Diversification of Cylindraspis commenced in the mid-Oligocene, long before the formation of the Mascarene Islands. This rejects any notion suggesting that the group either arrived from nearby or distant continents over the course of the last millions of years or had even been translocated to the islands by humans. Instead, Cylindraspis likely originated on now submerged islands of the Réunion Hotspot and utilized these to island hop to reach the Mascarenes. The final diversification took place both before and after the arrival on the Mascarenes. With Cylindraspis a deeply divergent clade of tortoises became extinct that evolved long before the dodo or the Rodrigues solitaire, two other charismatic species of the lost Mascarene fauna.

In quest of contact: phylogeography of helmeted terrapins (Pelomedusa galeata, P. subrufa sensu stricto).

Based on rangewide sampling and three mitochondrial and two nuclear markers (together up to 1,850 bp and 1,840 bp, respectively), we examine the phylogeography of two helmeted terrapin species (Pelomedusa galeata and P. subrufa sensu stricto) and infer shifts of climatically suitable spaces since the Last Glacial Maximum using a modeling approach. Whilst P. galeata displays significant phylogeographic structuring across its range and consists of two deeply divergent lineages that could represent distinct species, P. subrufa shows no obvious phylogeographic differentiation. This seems to be related to historically stable or fluctuating ranges. One of the lineages within P. galeata appears to be confined to the westernmost, winter-rainfall region of South Africa and deserves special conservational attention due to the scarcity of surface water. The other lineage is distributed further east and is differentiated in three weakly supported subclades with parapatric distribution; one occurring inland, and two along the south and east coasts, respectively. As far as is known, P. subrufa occurs in South Africa only in the northeast of the country (Limpopo, Mpumalanga) and we report the species for the first time from the Lapalala Wilderness Area in the Waterberg region (Limpopo), approximately 350 km further west than previously recorded. We confirmed the occurrence of P. galeata only 80 km south of Lapalala. Thus, a sympatric occurrence of P. galeata and P. subrufa is possible. Another putative contact zone, for the two lineages within P. galeata, must be located in the Western Cape region, and further contact zones are likely for the eastern subclades within P. galeata. The nuclear loci provided no evidence for gene flow across taxa or genetic clusters within taxa. Future investigations should use denser sampling from putative contact zones and more nuclear markers to re-examine this situation. Despite few phylogeographic studies published for southern African biota, it seems likely that differentiation follows general rules, and that climate and physiographic barriers (e.g., the Great Escarpment) have shaped phylogeographic patterns.

Disentangling the Pelomedusa complex using type specimens and historical DNA (Testudines: Pelomedusidae).

Recent research has shown that the helmeted terrapin (Pelomedusa subrufa), a species that occurs throughout sub-Saharan Africa, in Madagascar and the southwestern Arabian Peninsula, consists of several deeply divergent genetic lineages. Here we examine all nominal taxa currently synonymized with Pelomedusa subrufa (Bonnaterre, 1789) and provide mitochondrial DNA sequences of type specimens or topotypic material for most taxa. Lectotypes are designated for Testudo galeata Schoepff, 1792, Pentonyx capensis Duméril & Bibron, 1835, Pelomedusa nigra Gray, 1863, Pelomedusa galeata var. disjuncta Vaillant & Grandidier, 1910, and Pelomedusa galeata damarensis Hewitt, 1935. For Pelomedusa gasconi Rochebrune, 1884, a taxon without preserved type material, a neotype is designated. Type material of Pentonix americana Cornalia, 1849, a nominal species without credible type locality, is lost and its identity remains questionable. Also the holotype of Pelomedusa galeata orangensis Hewitt, 1935 is lost, but its allocation to the only genetic lineage occurring in South Africa is unambiguous. Phylogenetic analyses of type sequences or topotypic material reveal that the remaining nominal taxa represent three of the nine previously identified lineages of Pelomedusa. Among these three lineages is the South African one. Type specimens of Pentonyx gehafie Rüppell, 1835 correspond to an additional distinct lineage. The present study provides a sound basis for a subsequent integrative taxonomic revision of the Pelomedusa complex.

A revision of African helmeted terrapins (Testudines: Pelomedusidae: Pelomedusa), with descriptions of six new species.

Using nearly range-wide sampling, we analyze up to 1848 bp of mitochondrial DNA of 183             helmeted terrapins and identify a minimum of 12 deeply divergent species-level clades. Uncorrected p distances of these clades equal or clearly exceed those between the currently recognized species of Pelusios, the genus most closely related to Pelomedusa. We correlate genetic discontinuities of Pelomedusa with data on morphology and endoparasites and describe six new Pelomedusa species. Moreover, we restrict the name Pelomedusa subrufa (Bonnaterre, 1789) to one genetic lineage and resurrect three further species from its synonymy, namely P. galeata (Schoepff, 1792), P. gehafie (Rüppell, 1835), and P. olivacea (Schweigger, 1812). In addition to these ten Pelomedusa species, we identify two further clades from Cameroon and Sudan with similar levels of genetic divergence that remain unnamed candidate species. We also note that some problematical terrapins from South Africa and Somalia may represent two additional candidate species. Some of the Pelomedusa species are morphologically distinctive, whilst others can only be identified by molecular markers and are therefore morphologically cryptic taxa.

Deep genealogical lineages in the widely distributed African helmeted terrapin: evidence from mitochondrial and nuclear DNA (Testudines: Pelomedusidae: Pelomedusa subrufa).

We investigated the phylogeographic differentiation of the widely distributed African helmeted terrapin Pelomedusa subrufa based on 1503 base pairs of mitochondrial DNA (partial cyt b and ND4 genes with adjacent tRNAs) and 1937 bp of nuclear DNA (partial Rag1, Rag2, R35 genes). Congruent among different analyses, nine strongly divergent mitochondrial clades were found, representing three major geographical groupings: (1) A northern group which includes clades I from Cameroon, II from Ghana and Ivory Coast, III from Benin, Burkina Faso and Niger, IV from the Central African Republic, and V from Kenya, (2) a northeastern group consisting of clades VI from Somalia, and VII from Saudi Arabia and Yemen, and (3) a southern group comprising clade VIII from Botswana, the Democratic Republic of Congo, Madagascar and Malawi, and clade IX from South Africa. Malagasy and continental African populations were not clearly differentiated, indicating very recent arrival or introduction of Pelomedusa in Madagascar. The southern group was in some phylogenetic analyses sister to Pelusios, rendering Pelomedusa paraphyletic with respect to that genus. However, using partitioned Bayesian analyses and sequence data of the three nuclear genes, Pelomedusa was monophyletic, suggesting that its mitochondrial paraphyly is due to either ancient introgressive hybridization or phylogenetic noise. Otherwise, nuclear sequence data recovered a lower level of divergence, but corroborated the general differentiation pattern of Pelomedusa as revealed by mtDNA. This, and the depth of the divergences between clades, indicates ancient differentiation. The divergences observed fall within, and in part exceed considerably, the differentiation typically occurring among chelonian species. To test whether Pelomedusa is best considered a single species composed of deep genealogical lineages, or a complex of up to nine distinct species, we suggest a future taxonomic revision that should (1) extend the geographical sampling of molecular data, specifically focusing on contact zones and the possible sympatric occurrence of lineages without admixture, and (2) evaluate the morphology of the various genealogical lineages using the type specimens or topotypical material of the numerous junior synonyms of P. subrufa.

Living with the genetic signature of Miocene induced change: evidence from the phylogeographic structure of the endemic angulate tortoise Chersina angulata.

The phylogeographic structure of the monotypic endemic southern African angulate tortoise Chersina angulata was investigated throughout its distribution with the use of partial sequences from three mtDNA loci (COI, cyt b and ND4). Phylogeographic and phylogenetic structuring obtained for the three mtDNA markers were highly congruent and suggested the presence of two genetically distinct, reciprocally monophyletic evolutionary lineages. Group one contained two subclades with haplotypes from the north-western Cape and south-western Cape, respectively, while haplotypes from the southern Cape comprised group two. The two major clades were separated by nine and eight mutational steps for COI and ND4, respectively. Of the three mtDNA gene regions examined, the ND4 partial sequence contained the most phylogenetic signal. Haplotype diversity was generally low and we recovered 34 haplotypes for the 125 animals sequenced for the ND4 subunit. Nested clade analyses performed on the variable ND4 partial sequences suggested the presence of two major refugial areas for this species. The demographic history of the taxon was characterised by range expansion and prolonged historical fragmentation. Divergence time estimates suggest that the temporal and spatial distribution of the taxon was sculpted by changes in temperature and rainfall patterns since the late Miocene. Corroborative evidence from other reptiles is also suggestive of a late Miocene divergence, indicating that this was a major epoch for cladogenesis in southern Africa. Apart from the genetic differences between the two major clades, we also note morphometric and behavioural differences, alluding to the presence of two putative taxa nested within C. angulata.

Growing and shrinking in the smallest tortoise, Homopus signatus signatus: the importance of rain.

Climate change models predict that the range of the world's smallest tortoise, Homopus signatus signatus, will aridify and contract in the next decades. To evaluate the effects of annual variation in rainfall on the growth of H. s. signatus, we recorded annual growth rates of wild individuals from spring 2000 to spring 2004. Juveniles grew faster than did adults, and females grew faster than did males. Growth correlated strongly with the amount of rain that fell during the time just before and within the growth periods. Growth rates were lowest in 2002-2003, when almost no rain fell between September 2002 and August 2003. In this period, more than 54% of the tortoises had negative growth rates for their straight carapace length (SCL), shell height (SH), and shell volume (SV); maximum shrinking for SCL, SH, and SV was 4, 11, and 12%, respectively. The shell of H. s. signatus has some flexibility dorso-ventrally, so a reduction in internal matter due to starvation or dehydration may have caused SH to shrink. Because the length and width of the shell seem more rigid, reversible bone resorption may have contributed to shrinkage, particularly of the shell width and plastron length. Based on growth rates for all years, female H. s. signatus need 11-12 years to mature, approximately twice as long as would be expected allometrically for such a small species. However, if aridification lowers average growth rates to the level of 2002-2003, females would require 30 years to mature. Additionally, aridification would lower average and maximum female size, resulting in smaller eggs and hatchlings. These projected life history responses to aridification heighten the threat posed by the predicted range contraction of this red-listed species.

Parasites of domestic and wild animals in South Africa. XLVII. Ticks of tortoises and other reptiles.

A total of 586 reptiles, belonging to 35 species and five subspecies, were examined in surveys aimed at determining the species spectrum and geographic distribution of ticks that infest them. Of these reptiles 509 were tortoises, 28 monitor or other lizards, and 49 snakes. Nine ixodid tick species, of which seven belonged to the genus Amblyomma, and one argasid tick, Ornithodoros compactus were recovered. Seven of the ten tick species are parasites of reptiles. Amongst these seven species Amblyomma marmoreum was most prevalent and numerous on leopard tortoises, Geochelone pardalis; Amblyomma nuttalliwas present only on Bell's hinged tortoises, Kinixys belliana; and most Amblyomma sylvaticum were collected from angulate tortoises, Chersina angulata. Amblyomma exornatum (formerly Aponomma exornatum) was only recovered from monitor lizards, Varanus spp.; most Amblyomma latum (formerly Aponomma latum) were from snakes; and a single nymph of Amblyomma transversale (formerly Aponomma transversale) was collected from a southern African python, Python natalensis. All 30 Namaqualand speckled padloper tortoises, Homopus signatus signatus, examined were infested with O. compactus. The seasonal occurrence of A. sylvaticum and the geographic distribution of this tick and of A. marmoreum, A. nuttalli, A. exornatum, A. latum and O. compactus are illustrated.

Circadian rhythm of locomotor activity in the four-striped field mouse, Rhabdomys pumilio: a diurnal African rodent.

Although humans are diurnal in behaviour, animal models used for the study of circadian rhythms are mainly restricted to nocturnal rodents. This study focussed on the circadian behaviour of a rodent from South Africa that has a preference for daylight, the four-striped field mouse, Rhabdomys pumilio. In order to characterise the behavioural pattern of daily activity, locomotor rhythms were studied under different light regimes using an automated data recording system. Under conditions of natural daylight, which include dawn and dusk transitions, R. pumilio exhibited activity restricted to the daytime period. Activity was concentrated around morning and evening with a decrease during mid-day. A similar diurnal preference pattern of behaviour was recorded under a light-dark cycle of artificial illumination. Under conditions of constant darkness, the four-striped field mouse exhibited a free-running circadian rhythm of locomotor activity with activity concentrated during the subjective day. Free-running rhythms varied greatly between individuals, from slightly less to slightly more than 24 h (range = 23.10 to 24.80 h). Under conditions of constant light, the mice were more active during subjective day, but the free-running rhythm in all individuals was consistently longer than 24 h (range = 24.30 to 24.79 h).

Viewing chelonian reproductive ecology through acoustic windows: cranial and inguinal perspectives.

Ultrasonography is a powerful new tool in reproductive physiology and ecology but quantitative evaluations of acoustic window efficacies are necessary to assess methodological accuracy. We evaluated the efficacy of the left inguinal, right inguinal and cranial acoustic windows for detecting ovarian follicles and oviducal eggs in three tortoise species. Incorporating cranial results improved upon inguinal efficacies for detecting follicles in the three species, and for detecting eggs in Chersina angulata, which had small inguinal apertures for its body size. The cranial window provided the best follicle results (highest numbers detected) but the left inguinal window provided the best egg results. This asymmetry was probably due to the primarily posterior position of the oviducts (eggs) and the pliable ovarian sheets allowing follicles to move cranially. The left-right asymmetry may result from intestinal asymmetry (i.e., between the cecum and descending colon) in obscuring ultrasound transmission or displacing follicles and eggs. Seasonal influences upon efficacies varied with species, partially due to species differences in a) reproductive cycles and b) size and number of follicles and eggs. We attributed other efficacy differences among species to differences in body size and shell morphology, especially the inguinal apertures. Correlation analyses revealed strong individual influences upon window efficacy, suggesting that individuals varied in the position of follicles and eggs and the degree of obfuscation by nonreproductive structures. The paucity of correlations between efficacies and body size reflects the complex interactions of body size, shell morphology, reproductive structures and reproductive cycles on efficacies.