Barcoding,molecular taxonomy,and exploration of the diversity of shrews (Soricomorpha: Soricidae) on Mount Nimba (Guinea)

We tested the efficiency of cytochrome oxidase I (COI)‐barcoding as a taxonomic tool to discriminate and identify sympatric shrew species on Mount Nimba (Guinea). We identified 148 specimens at the species level using morphological characters and comparison with type specimens, including several taxa from Mount Nimba. We identified ten morphospecies and tested aspects of genetic diversity and monophyly using genetic data from three mitochondrial (16S, cytochrome b, and COI) and one nuclear marker (the breast cancer gene, BRCA). Nine morphospecies were validated under the phylogenetic and genetic species concepts, including the recently diverged species Crocidura buettikoferi, Crocidura theresae, and Crocidura grandiceps. Under the same concepts, our analyses revealed the presence of two cryptic species amongst animals identified as Crocidura muricauda. We then tested the efficiency of barcoding thanks to commonly used phenetic methods, with the 148 specimens representing 11 potentially valid species based on morphological and molecular data. We show that COI‐barcoding is a powerful tool for shrew identification and can be used for taxonomic surveys. The comparison of genetic divergence values shows the presence of a barcoding gap (i.e. difference between the highest intraspecific and the lowest interspecific genetic divergence values). Given that only a few COI sequences are available for Afrotropical shrews, our work is an important step forward toward their enrichment. We also tested the efficiency of the three other sequenced markers and found that cytochrome b is as efficient as COI for barcoding shrews. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166 , 672–687.     

Bones and genes: resolution problems in three Vietnamese species of Crocidura (Mammalia,Soricomorpha, Soricidae) and the description of?an?additional new species

Recent investigations of Southeast Asian white toothed shrews belonging to the genus Crocidura have revealed discrepancies between the results of morphological and molecular studies. The following study concerns three species of Crocidura occurring in Vietnam, namely Crocidura attenuata, Crocidura tanakae and Crocidura wuchihensis, and an undescribed fourth species revealed by molecular analysis. For many years Crocidura attenuata has been known to occur in Vietnam but, until very recently, the morphologically similar and comparably sized Crocidura tanakae was believed to be restricted to Taiwan. Following several molecular studies over the last few years, this species is now believed to be considerably more widespread and recognised as occuring also in Vietnam. The results of one of these recent molecular studies also revealed the presence of an undescribed species of Crocidura, similar in size and morphology to Crocidura wuchihensis, which is herein described. Data are provided on geographical variation in Vietnam and the problems of defining morphologically similar yet molecularly disparate species are discussed.     

Phylogeny,phylogeography and geographical variation in the Crocidura monax (Soricidae) species complex from the montane islands of Tanzania,with descriptions of three new species

We assess morphological and multilocus genetic variation among 11 isolated montane populations of white‐toothed shrews from Tanzania that have been referred to either Crocidura monax Thomas or C. montis Thomas. The montane sites we sampled represent ‘sky‐islands’ from two geologically distinct archipelagos (Northern Highlands and the Eastern Arc Mountains) and are a significant component of the Eastern Afromontane Biodiversity Hotspot. We used multivariate analyses of morphometric traits and phylogenetic and species‐delimitation analyses of multilocus DNA sequence data to assess species‐level diversity. Our species delimitation analyses included a novel, pairwise validation approach that avoids potential biases associated with specifying a guide tree. These analyses reveal several distinct lineages, which we treat as six allopatric species. Each species is restricted to one, two or four mountains. We use available names to recognize C. monax, C. tansaniana Hutterer and C. usambarae Dippenaar, while naming and describing three new species. Our results demonstrate the effectiveness of combining morphological and genetic data to uncover and describe hidden diversity in a cryptic mammalian system. © 2015 The Linnean Society of London     

Molecular taxonomy of Crocidura species (Eulipotyphla: Soricidae) in a key biogeographical region for African shrews,Nigeria

The taxonomy of African shrew species is still unresolved due to their conserved morphology. This also affects knowledge concerning their geographic distribution. In Nigeria, using mitochondrial Cytochrome b gene sequences, we carried out a survey for shrews from the genus Crocidura across various ecological zones to determine taxa that are present and also to assess their phylogeographic structure. Our analyses include 183 specimens collected with Sherman traps from 19 localities around the country. We detected six taxa: Crocidura olivieri lineages II, III and IV, C. hildegardeae, C. jouvenetae, and C. foxi. Among these, C. hildegardeae and C. jouvenetae are reported in Nigeria for the first time. Phylogenetic comparison of our genetic sequences to those generated from other parts of Africa demonstrate that all species in our study, as currently defined, are in need of taxonomic revision. Geographically, Nigeria seems to represent the easternmost boundary for C. olivieri lineage II and C. jouvenetae, and the western distribution limit of C. olivieri lineage IV and C. hildegardeae. The Niger River appears to be the most significant topographical barrier restricting these taxa. This information is vital to preserving the diversity but also managing the epidemiological potential of these small mammals.     

Declining body size in an endangered large mammal

Body size affects almost every aspect of the biology of a species. According to the ‘resource rule’, decreasing resource availability (e.g. prey density) will lead to a reduction in body size or, alternatively, a decline in mass‐independent energy expenditure. In the present study, we provide a test of this hypothesis, assessing the effect of significantly decreasing prey density on endangered African wild dog (Lycaon pictus) body size and energy expenditure over a 20‐year period. As predicted from the ‘resource rule’, decreasing resource availability resulted in energetic re‐allocation: wild dogs' body size decreased significantly (both shorter and slimmer), whereas our fitness‐related measure of energy expenditure (i.e. litter size) remained constant over time. A phenotypic change of up to 17% within 20 years, as found in the present study, appears to be unprecedented in a nonharvested large mammal, thus advancing the emerging field of eco‐evolutionary dynamics. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 8–12.     

Phylogenetic relationships and speciation patterns in an African savanna dwelling bird genus (Myrmecocichla)

Phylogenetic relationships among eight of nine Myrmecocichla chat species were inferred from DNA data. Bayesian posterior probabilities and maximum‐likelihood bootstrap percentages strongly supported most branches in the phylogeny. Based on these results, Myrmecocichla, as currently defined, is not monophyletic. The results indicated that Myrmecocichla albifrons is part of a Cercomela + Oenanthe clade, whereas Oenanthe monticola is shown to be a Myrmecocichla. In addition, Myrmecocichla arnotti is shown to be polyphyletic. Phylogenetic analyses support three Southern versus Eastern or Northern speciation events. The dating of these speciation events suggests that they correspond to periods when the Afrotropical forests were expanded to coastal Kenya, 3–5 Mya. This forest expansion thus served as a vicariant driver of speciation in the genus, a result consistent with speciation patterns in other arid‐adapted African bird genera. Our haplotype analysis within one of the most widespread and habitat diverse Myrmecocichla species (formicivora, a southern African endemic) showed little genetic variation. Along with speciation patterns shown for Myrmecocichla and other avian genera, this lack of standing variation would appear to support large, inter‐regional drivers of speciation as having the largest effect on the diversification of arid‐adapted Africa bird species, which is in stark contrast to other vertebrate lineages whose genetic structure often shows strong intra‐regional effects. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 180–190.     

How common is gigantism in insular fossil shrews? Examining the ‘Island Rule’ in soricids (Mammalia: Soricomorpha) from Mediterranean Islands using new body mass estimation models

The evolution of organismal body size in extant and extinct ecosystems of islands (Island Rule) is receiving much attention at present. Allometric models are a reliable way to predict the weight of extinct species, but are scarce or even absent for some groups of micromammals. To fill the gap, we carried out regression models with extant species of soricids (N = 63) using measurements of teeth, cranium, and postcranial bones, and applied these to fossil insular species and their mainland ancestors. Almost all models are significant (P < 0.05), except for those based on the width of occipital condyles. The femur can be considered the most reliable body‐mass predictor, producing estimations not far from those derived from teeth (excepting molar widths). Predictions of insular species (in grams) show that those belonging to the tribe Nectogalini [Asoriculus burgioi Masini & Sarà, 1998 , 27.54; Asoriculus similis (Hensel, 1855), 23.68; Nesiotites ponsi Reumer, 1979, 14.58; Nesiotites meloussae Pons‐Moyà & Moyà‐Solà, 1980, 24.83; Nesiotites hidalgo Bate, 1945, 26–30] had larger masses than Crocidura sp. [Crocidura sicula esuae (Kotsakis, 1986), 9.50; Crocidura sicula sicula (Miller, 1901), 8.60; Crocidura zimmermanni Wettstein, 1953, 7–10]. Statistical comparisons with their ancestors revealed that certain species (Nesiotites sp. from Mallorca and A. similis from Sardinia) may be considered giants, but not C. zimmermanni (from Crete). Body size is closely related to life history, which is highly influenced by the selective regimes of the environment. Thus, the lower isolation distance of Crete in comparison with Sardinia and Mallorca, suggesting more introductions of competitors and predators, and the presence of a flow with the mainland, may be the reason for the absence of a giant form of C. zimmermanni. However, some biological aspects of species (such as phylogeny or lifestyle) may also have an influential role.     

Forest refugia and riverine barriers promote diversification in the West African pygmy shrew (Crocidura obscurior complex,Soricomorpha)

The Crocidura obscurior or West African pygmy shrew complex is endemic to West African forests from south‐eastern Guinea, eastern Liberia, southern Côte d'Ivoire and south‐western Ghana. We explore the genetic and morphometric diversity of 239 individuals of the C. obscurior complex from 17 localities across its geographical range. Using genetic data from three mitochondrial (16S, cytochrome b and COI) and four nuclear markers (BRCA1, STAT5A, HDAC2 and RIOK3) and skull geometric morphometrics, we show that this complex is composed of two cryptic and sympatric species, C. obscurior and C. eburnea. We then test several hypotheses to infer their evolutionary history. The observed phylogeographical pattern based on cytochrome b and COI sequences fits the forest refuge theory: during arid phases of the Plio‐Pleistocene, around 3.5, 2.1, 1 and 0.5 Mya, a small number of populations survived in isolated forest patches and diverged allopatrically. During wetter climatic periods, forests expanded, leading to secondary contacts between previously isolated populations. Our results also suggest the possible contribution of episodes of isolation in subrefuges. Historical variation of the West African hydrographic network could also have contributed to the observed patterns of genetic differentiation. Rivers such as the Volta and Sassandra may act as past and/or current barriers to gene flow. Although these two species have sympatric distributions, their phylogeographical histories are somewhat dissimilar due to small differences in their dispersal abilities and ecological requirements.     

Carving out turf in a biodiversity hotspot: multiple,previously unrecognized shrew species co‐occur on Java Island,Indonesia

In theory, competition among species in a shared habitat results in niche separation. In the case of small recondite mammals such as shrews, little is known about their autecologies, leaving open questions regarding the degree to which closely related species co‐occur and how or whether ecological niches are partitioned. The extent to which species are able to coexist may depend on the degree to which they exploit different features of their habitat, which may in turn influence our ability to recognize them as species. We explored these issues in a biodiversity hotspot, by surveying shrew (genus Crocidura) diversity on the Indonesian island of Java. We sequenced portions of nine unlinked genes in 100–117 specimens of Javan shrews and incorporated homologous data from most known Crocidura species from other parts of island South‐East Asia. Current taxonomy recognizes four Crocidura species on Java, including two endemics. However, our phylogenetic, population genetic and species delimitation analyses identify five species on the island, and all are endemic to Java. While the individual ranges of these species may not overlap in their entirety, we found up to four species living syntopically and all five species co‐occurring on one mountain. Differences in species' body size, use of above ground‐level habitats by one species and habitat partitioning along ecological gradients may have facilitated species diversification and coexistence.     

Record of five new endemic small mammal hosts for four ectoparasite species from Sri Lanka

Both ticks and fleas are medically important arthropods, which act as vectors for a number of zoonotic diseases. In Sri Lanka, 35 species of ticks and 20 species of fleas have been reported from mammals, birds and reptiles. However, records of these parasites from small mammals are scanty. Here, we report ticks and fleas infesting muroid rodents and shrews including endemic species collected from around the country. Of the 149 small mammals collected, 48 individuals were infested with ticks, fleas or both. Immature stages of three tick species, Ixodes ceylonensis, Haemaphysalis hystricis and Hyalomma brevipunctata and unidentified species of Rhipicephalus were recorded from ten small mammal species. Four species of fleas, Stivalius phoberus, Stivalius aporus, Lentistivalius ferinus and Nosopsyllus tamilanus were identified from five species of small mammals. We report new host species for I. ceylonensis (Crocidura miya, Suncus montanus and Solisorex pearsoni), S. aporus (S. montanus and Mus fernandoni), N. tamilanus (S. pearsoni) and Rhipicephalus spp. (Crocidura hikmiya, S. montanus, S. pearsoni, M. fernandoni). All new host species reported here are endangered or critically endangered endemics to Sri Lanka.     

Phylogeny and species delimitation of the C‐genome diploid species in Oryza

Abstract The diploid Oryza species with C‐genome type possesses abundant genes useful for rice improvement and provides parental donors of many tetraploid species with the C‐genome (BBCC, CCDD). Despite extensive studies, the phylogenetic relationship among the C‐genome species and the taxonomic status of some taxa remain controversial. In this study, we reconstructed the phylogeny of three diploid species with C‐genome (Oryza officinalis, O. rhizomatis, and O. eichingeri) based on sequences of 68 nuclear single‐copy genes. We obtained a fully resolved phylogenetic tree, clearly indicating the sister relationship of O. officinalis and O. rhizomatis, with O. eichingeri being the more divergent lineage. Incongruent phylogenies of the C‐genome species found in previous studies might result from lineage sorting, introgression/hybridization and limited number of genetic markers used. We further applied a recently developed Bayesian species delimitation method to investigate the species status of the Sri Lankan and African O. eichingeri. Analyses of two datasets (68 genes with a single sample, and 10 genes with multiple samples) support the distinct species status of the Sri Lankan and African O. eichingeri. In addition, we evaluated the impact of the number of sampled individuals and loci on species delimitation. Our simulation suggests that sampling multiple individuals is critically important for species delimitation, particularly for closely related species.     

Phylogenetic evolution and biogeography of Southeast Asian shrews (genus Crocidura: Soricidae)

Genetic variation of 20 species of shrews from the Malay peninsula, Sumatra, Java, Borneo, the Philippines and Sulawesi was assessed by allozyme electrophoresis at 32 loci. According to Mantel's tests, the genetic differentiation of these species of shrews is not a function of the geographic distance separating them (r = 0.09, NS), but is correlated to the water depth surrounding the islands where they live (r = 0.49, P < 0.01). The results are just the reverse if the correlations are computed for the Sunda Shelf taxa only. In this case, the sampled populations show an isolation-by-distance relationship (r = 0.32; P < 0.01), while no significant correlation with water depth was detected (r = 0.20; P - 0.07). Qualitative predictions based on eustatic sea level variation and water depth were formulated as a model of historic connections between the islands. This palaeogeographic model was tested through Brooks Parsimony Analysis. The assumption of a simple vicariant evolution of the shrews was rejected, but several concordant patterns indicate that the phytogeny of these mammals was indeed shaped by these events. Homoplasies demonstrated that the SE Asian species of Crocidura include composite zoogeographic histories. Sulawesi, for example, supports at least six species, five of which are closely related, while the last one, C. nigripes, is more closely related to a Bornean taxon. This pattern was interpreted as the result of a first wave of colonizers which subsequently radiated, followed by a more recent, second colonization event from Borneo. The overall small genetic distance found within the assemblage of the five old endemics (D_N= 0.151 pL 0.041) suggests that the radiation was not accompanied by extensive differentiation, although from a karyological point of view, they exhibit unusual variations when compared to other Indomalayan Crocidura. By contrast, the four species found on Sumatra are more differentiated (D_N= 0.221 pL 0.063) and never form sister-group relationships in any phylogenetic reconstruction; each one is more closely related to different taxa living outside Sumatra. This suggests that they are probably remnants of an important centre of dispersal for the entire Malay Archipelago. The standard genetic distance averaged among all Southeast Asian species (D_N= 0.235 pL 0.094) is about half that measured within Palearctic or African taxa. Such an overall lower mean level of genetic variability is consistent with the hypothesis of a relatively recent colonization of the Malay Archipelago by shrews of the genus Crocidura.     

Local endemism and within‐island diversification of shrews illustrate the importance of speciation in building Sundaland mammal diversity

Island systems are important models for evolutionary biology because they provide convenient, discrete biogeographic units of study. Continental islands with a history of intermittent dry land connections confound the discrete definitions of islands and have led zoologists to predict (i) little differentiation of terrestrial organisms among continental shelf islands and (ii) extinction, rather than speciation, to be the main cause of differences in community composition among islands. However, few continental island systems have been subjected to well‐sampled phylogeographic studies, leaving these biogeographic assumptions of connectivity largely untested. We analysed nine unlinked loci from shrews of the genus Crocidura from seven mountains and two lowland localities on the Sundaic continental shelf islands of Sumatra and Java. Coalescent species delimitation strongly supported all currently recognized Crocidura species from Sumatra (six species) and Java (five species), as well as one undescribed species endemic to each island. We find that nearly all species of Crocidura in the region are endemic to a single island and several of these have their closest relative(s) on the same island. Intra‐island genetic divergence among allopatric, conspecific populations is often substantial, perhaps indicating species‐level diversity remains underestimated. One recent (Pleistocene) speciation event generated two morphologically distinct, syntopic species on Java, further highlighting the prevalence of within‐island diversification. Our results suggest that both between‐ and within‐island speciation processes generated local endemism in Sundaland, supplementing the traditional view that the region's fauna is relictual and primarily governed by extinction.     

Failure of sexual reproduction found in micropropagated critically endangered plants prior to reintroduction: a cautionary tale

Micropropagation is a useful technique for ex situ multiplication and restoration of critically endangered plant species, but the sexual reproductive behaviour of micropropagated plants is seldom evaluated prior to reintroduction. We examined the critically endangered species Rulingia sp. ‘Trigwell Bridge’, with only three remaining plants known in the wild, as a model case to examine this issue. Abnormalities in micropropagated plants of this species related to four floral traits (lengths of sepals, petals and anthers and width of anthers). The number of pollen grains per flower of abnormal individuals was lower than in plants with apparently normal flowers (wild types), but not significantly so (P = 0.068). Pollen viability for the abnormal plant (0.87 ± 0.26%) was significantly lower than for the plants exhibiting wild‐type floral morphology (45.42 ± 4.47%). Experimental manipulations were used to examine the mating behaviour of normal and abnormal plants. The results showed that both male and female reproductive failure was linked to individuals exhibiting abnormal flowering attributes. Such aberrant reproductive performance in a micropropagated rare species predicates caution when using micropropagated plants in reintroduction programmes, highlighting the importance of screening for reproductive normality prior to release of micropropagated plants (especially for critically endangered species where reliance on in vitro propagation methods is often a necessity). © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 165 , 278–284.     

From the Neotropics to the Namib: evidence for rapid ecological divergence following extreme long‐distance dispersal

Extreme long‐distance dispersal is an important process in plant biogeography. Such events can lead to rapid diversification due to founder effects, genetic drift and novel selection in recipient environments. Balloon vines (Cardiospermum spp.) are mainly Neotropical, but include two native southern African species, the endemic desert‐adapted C. pechuelii and the moist subtropical C. corindum (which also occurs in the Neotropics). We used phylogenetic approaches (internal transcribed spacer (ITS), rpl32 and trnL‐trnF DNA sequencing data) and population genetics (amplified fragment length polymorphism (AFLP) analyses) to confirm the long‐distance dispersal of C. corindum to southern Africa and to reveal the subsequent divergence of the morphologically and ecologically extreme but genetically close C. pechuelii. We could not judge whether incongruences between ecological requirements and morphology and gene trees for the African species resulted from ongoing gene flow or incomplete lineage sorting, but our findings do support recent divergence of C. pechuelii from C. corindum in Africa following transoceanic dispersal of the lineage. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 477–486.     

Local genetic structure in the critically endangered,cave‐associated perennial herb Primulina tabacum (Gesneriaceae)

The local spatial genetic structures of cave‐associated plants are seldom studied. Given that these plants are mainly confined to small areas in and around the entrances of caves, we hypothesized that they might lack genetic structures at local scales. To test this hypothesis, we sampled two large populations (named D and T) of a critically endangered perennial herb, Primulina tabacum, which is endemic to karst caves in southern China. We analysed nine microsatellite loci and sequenced four chloroplast DNA (cpDNA) intergenic spacer regions to study the genetic diversity and structure within and between both populations. Both populations have distinct genetic characteristics. Samples from two subpopulations in population D showed considerable genetic divergence. This is not consistent with the hypothesis that P. tabacum has a weak genetic structure at a local scale. However, 94% of the individuals in population T shared the same multilocus genotype, which indicates little genetic structure within this population. The contributions of seed flow, pollen flow and (sub)population history to the genetic diversity and structure in each and both populations are discussed. Our study is the first to investigate local genetic diversity and structure in a cave‐associated plant, and provides valuable information for the sustainable conservation of such species. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 747–756.     

Three new species of Callulina (Amphibia: Anura: Brevicipitidae) highlight local endemism and conservation plight of Africa's Eastern Arc forests

Material ascribed to the genus Callulina from north‐east Tanzania and south‐east Kenya is assessed. Three new species of Callulina are described from the North ( Callulina laphami sp. nov. ) and South ( Callulina shengena sp. nov. and Callulina stanleyi sp. nov. ) Pare Mountains in Tanzania. The species are diagnosed based on morphological, acoustic, and molecular data. A new key to the species of Callulina is provided. Based on an interpretation of the International Union for Conservation of Nature (IUCN) red list, we suggest that the three species will qualify as critically endangered, because of their small distributions and the ongoing threat to their habitat. We reveal the high local endemism of Callulina in the northern part of the Eastern Arc Mountains, with each species restricted to no more than one mountain (fragment) block. This high local endemism in Callulina is probably widespread across the Eastern Arc, raising further conservation concern for this group of amphibians. Based on new molecular phylogenetic data for Callulina, we discuss biogeographical relationships among north‐east Tanzanian mountains, and evolutionary patterns in Eastern Arc breviciptids. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160 , 496–514.     

‘After Africa’: the evolutionary history and systematics of the genus Charaxes Ochsenheimer (Lepidoptera: Nymphalidae) in the Indo‐Pacific region

The predominantly Afrotropical genus Charaxes is represented by 31 known species outside of Africa (excluding subgenus Polyura Billberg). We explored the biogeographic history of the genus using every known non‐African species, with several African species as outgroup taxa. A phylogenetic hypothesis is proposed, based on molecular characters of the mitochondrial genes cytochrome oxidase subunit I (COI) and NADH dehydrogenase 5 (ND5), and the nuclear wingless gene. Phylogenetic analyses based on maximum parsimony and Bayesian inference of the combined dataset implies that the Indo‐Pacific Charaxes form a monophyletic assemblage, with the exception of Charaxes solon Fabricius. Eight major lineages are recognized in the Indo‐Pacific, here designated the solon (+African), elwesi, harmodius, amycus, mars, eurialus, latona, nitebis, and bernardus clades. Species group relationships are concordant with morphology and, based on the phylogeny, we present the first systematic appraisal and classification of all non‐African species. A biogeographical analysis reveals that, after the genus originated in Africa, the evolutionary history of Charaxes in the Indo‐Pacific, in particular Wallacea, may be correlated with the inferred geological and climatic history of the region. We propose that Wallacea was the area of origin of all Charaxes (excluding C. solon) occurring to the east of Wallace's [1863] Line. The earliest Indo‐Pacific lineages appear to have diverged subsequent to the initial fragmentation of a palaeo‐continent approximately 13 million years ago. Further diversification in Indo‐Pacific Charaxes appears primarily related to climatic changes during the Pliocene and possibly as recently as the Pleistocene. Although both dispersal and vicariance have played important roles in the evolution of the genus within the region, the latter has been particularly responsible for diversification of Charaxes in Wallacea. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 457–481.