Genetic variation and population differentiation of Michelia formosana (Magnoliaceae) based on cpDNA variation and RAPD fingerprints: relevance to post-Pleistocene recolonization

We used sequence variation of the atpB- rbcL intergenic spacer of cpDNA and nested clade analysis to assess the phylogeographic pattern of Michelia formosana, a species restricted to Taiwan and the Ryukyus. In total, 31 haplotypes were identified and clustered into four major chlorotypes. Genetic composition of nearly all populations was heterogeneous and paraphyletic phylogenetically. Although the apportionment of cpDNA variation hardly revealed a geographic pattern due to the coancestry of dominant sequences, some chlorotypes were restrictedly distributed. According to the patterns of clade dispersion and displacement, a reconstructed minimum spanning network revealed that historical events of past fragmentation and range expansion, associated with glaciation, may have shaped the phylogeographic patterns of M. formosana. Four possible refugia were identified: the Iriomote and Ishigaki Islands (the southern Ryukyus), Wulai (northern Taiwan), and Nanjen (southern Taiwan), on the basis of the interior positions of their haplotypes in the network and the high level of nucleotide diversity. Given insufficient time for coalescence at the cpDNA locus since the late Pleistocene recolonization, lineage sorting led to low levels of genetic differentiation among populations. In contrast, hierarchical examination of the random amplified polymorphic DNA (RAPD) data scored from six populations across three geographical regions, using an analysis of molecular variance (AMOVA), indicated high genetic differentiation both among populations (Phi(ST) = 0.471) and among regions (Phi(CT) = 0.368). An unweighted pair group method with arithmetic mean (UPGMA) tree of the RAPD fingerprints revealed that populations of two offshore islands of eastern Taiwan ( M. formosana var. kotoensis) were clustered with geographically remote populations of the Ryukyus instead of those in southern Taiwan, suggesting some historical division due to geographic barriers of the central mountain range. In contrast to the paraphyly of the nearly neutral cpDNA alleles, differentiated RAPDs may have experienced diversifying selection.     

Protecting evolutionary significant units for the remnant populations of <Emphasis Type="Italic">Berchemiella wilsonii</Emphasis> var. <Emphasis Type="Italic">pubipetiolata</Emphasis> (Rhamnaceae)

Berchemiella wilsonii var. pubipetiolata (Rhamnaceae) is an endangered tree in eastern China. Habitat destruction has resulted in fragmentation of remnant populations and extinction of local populations. AFLP and cpDNA markers were used to determine the population structure of remnant populations of B. wilsonii var. pubipetiolata. Moderate nuclear genomic diversity was found within each of the four remnant populations (H _S = 0.141–0.172), while the cpDNA haplotype diversity in each population ranged from 0.356 to 0.681. Six haplotypes were identified by a combined cpRFLP and cpSSR analysis in a total of 89 individuals. AMOVA revealed significantly AFLP genetic differentiation within and between regions (Φ_SC = 0.196, Φ_CT = 0.396, respectively), and a high cpDNA haplotype differentiation between regions (Φ_CT = 0.849). The results suggest low gene flow between populations of B. wilsonii var. pubipetiolata. Strong genetic divergence between two regional populations as revealed by both AFLP and cpDNA markers provided convincing evidence that two distinct evolutionary lineages existed, and should be recognized as ‘evolutionary significant units’ (ESUs) for conservation concerns.     

Two genetic divergence centers revealed by chloroplastic DNA variation in populations of <Emphasis Type="Italic">Cinnamomum kanehirae</Emphasis> Hay

Cinnamomum kanehirae Hayata (Lauraceae), the most valuable subtropical and temperate broadleaf timber tree in Taiwan, is rapidly disappearing from the wild. Taking advantage of a scion garden established by the Taiwan Forestry Research Institute, we examined patterns of chloroplast DNA (cpDNA) variations in 19 populations including 94 individuals. By sequencing two cpDNA fragments using universal primers (the trnL-trnF and petG-trnP intergenic spacers), we found eight polymorphic sites, six haplotypes, and extremely low nucleotide diversity (π = 0.00016) from 792 bp aligned sequences. The ancestral haplotype is widely distributed. Among the populations studied, three separated populations, at Yungfeng, Fuli, and Tahu have high nucleotide diversity. No phylogeographical structures of haplotypes were revealed because the tests of N _ST−G _ST for populations did not differ from zero in any situations; a ‘star-like’ genealogy is characteristic when all haplotypes rapidly coalesce and is a general outcome of population expansion. The neutrality test also suggested demographic expansion. The genetic divergence and diversity analyses suggested that two potential refugia existed during the last glaciation with a major one located in southeastern Taiwan and a minor one located in Tahu in north-central Taiwan in the Hsuehshan Range, west of the Central Mountain Range.     

Phylogeography of Trochodendron aralioides (Trochodendraceae) in Taiwan and its adjacent areas

Aim This paper described current phylogeographical patterns of chloroplastic DNA variation of Trochodendron aralioides, a temperate tree species, and inferred its possible refugium in Taiwan. This information was compared with the known phylogeographical pattern of subtropical tree species. Location A total of 24 populations were sampled including 20 from Taiwan, two each from the Ryukyus and Japan. Methods A haplotype network was constructed by computer program TCS, various parameters of genetic diversity were calculated and neutrality was tested by computer program DnaSP. To examine the similarity of genetic structure among populations, a maximum parsimony tree was reconstructed by computer program PAUP*. The results of isozyme of T. aralioides from a previous publication were incorporated into this study to infer the phylogeographical history. Results Nine haplotypes according to six substitutions, two indels and one inversion of the two cpDNA intergenic spacer fragments (petG‐trnP and petA‐psbJ) of T. aralioides were recognized. Genetic structure of the population of Japan is totally different from those of Taiwan and the Ryukyus. In Taiwan, the genetic structure was differentiated among populations revealed by G_st = 0.700 and N_st = 0.542, and the population genetics was clearly spatially structured. Two population groups were recognized. The first group was distributed islandwide and extended to the Ryukyus. The second group contained five of the seven known haplotypes, and was restricted to the area between latitude 24°46′ and 24°06′ N. Conclusions In Taiwan, north‐central area between latitude 24°46′ and 24°06′ N is potentially a refugium during the last glaciations. This finding is contradicted to subtropical species as Cyclobalanopsis glauca.     

Contrasting phylogeographical patterns of two closely related species, Machilus thunbergii and Machilus kusanoi (Lauraceae), in Taiwan

Aim The purpose of this paper was to study the patterns of genetic variation, demographic history, haplotype relationships and potential location of diversity centres of two closely related species, Machilus thunbergii and Machilus kusanoi. Location The phylogeography of M. thunbergii and M. kusanoi was examined by sampling 110 and 106 individuals from 25 and 16 sampling sites, respectively, across their distributional range in Taiwan. Machilus thunbergii is distributed on the Asian mainland, South Korea, southern Japan, the Ryukyus, Taiwan and the Philippines, whereas M. kusanoi is endemic to Taiwan. These two species are closely related, and both are widely distributed in Taiwan but occupy different altitudinal zones and habitats. Methods The range‐wide variation of M. thunbergii and M. kusanoi in Taiwan was studied using chloroplast DNA (cpDNA) variations. A haplotype network was constructed with the computer program tcs . Nested clade analysis was conducted with the computer program ceodis , and various parameters of genetic diversity were calculated and neutrality tested by the computer program Dna SP. Population differentiation was estimated using the programs arlequin and hapstep . The contribution of the populations to gene diversity and to allelic richness was calculated using the software contrib . The level of divergence for each population from the remaining populations was calculated as the mean value of pairwise F_ST for each population against the rest of the populations. Results Extremely low levels of genetic differentiation were found for both species. This result suggested that these two species probably survived in multiple relict refugia with different population sizes throughout the island during low‐temperature periods of the Pleistocene. In addition, nested clade analysis (NCA) of cpDNA haplotypes indicated that restricted gene flow with isolation‐by‐distance characterized the recolonization after the Pleistocene by Tashueshan and Shiouhluan populations of M. thunbergii in the north‐central area west of the Central Mountain Range (CMR). In contrast, NCA analysis indicated that a major diversity centre on the southern tip of the island (Kending population) and contiguous range expansion characterized the recolonization by M. kusanoi of northern areas along the east side of the CMR. The major diversity centres found for the two species examined were further supported by the results of the mean F_ST for individual populations in comparison with other populations, and of the contribution of the divergence component to the total diversity. Main conclusions This research supports the multiple relict refugia hypothesis for both species investigated. Populations of M. thunbergii at Shiouhluan and Tashueshan in the north‐central area west of the CMR represent a diversity centre currently expanding its size. A diversity centre at the southern‐edge population of M. kusanoi, and a contiguous range expansion from Kending, were found. These results indicate that the M. thunbergii populations at Tashueshan and Shiouhluan and the M. kusanoi population at Kending, and even Soukar, are evolutionarily significant units for conservation programmes.     

Phylogeographical studies in disjunct populations of <Emphasis Type="Italic">Symplocos laurina</Emphasis> Wall. using cytoplasmic PCR-RFLP approach

Genetic variation, haplotype relationships, and potential regions of diversity in Symplocos laurina Wall. (Symplocaceae), a montane tree species from India, have been tested using molecular and biogeographical data to infer phylogeographic patterns. The polymerase chain reaction-restriction fragment length polymorphism technique was used to determine the chloroplast (cp) and mitochondrial (mt) DNA haplotypes of 218 individuals from 12 populations, collected from Northeast India (NEI), which is a part of Indo-Burma biodiversity hotspot, and South India, which includes the Western Ghats (WG), another biodiversity hotspot from India and Eastern Ghats (EG). Nine cpDNA (chlorotypes) and 24 mtDNA haplotypes (mitotypes) were identified; the WG region was identified as the most diverse for chlorotypes and the NEI region for mitotypes. Although a strong population differentiation was observed, phylogeographic structure was found to be absent for both the genomes. The haplotype network revealed the presence of two main lineages: NEI-WG lineage and EG lineage, largely without overlapping distributions. The study tests two hypotheses, namely vicariance and dispersal, to understand the distribution of plants in India. The population structure of S. laurina strongly suggests the persistence of the species in putative refugial areas preglaciation and further establishment of other populations of S. laurina from these refugial populations.     

Geographic distribution of chloroplast variation in Italian populations of beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.)

The distribution of chloroplast DNA (cpDNA) variation in Italian beech (Fagus sylvatica L.) populations was studied using PCR-RFLP and microsatellite markers. In total, 67 populations were analysed, and 14 haplotypes were identified by combining the two marker types. A remarkable subdivision of cpDNA diversity in Italian beech was found, as indicated by a high level of genetic differentiation (G_st=0.855). The highest level of total haplotype diversity (h_t=0.822) was estimated for southern Italian populations. The highest number of haplotypes was found in the central-southern region of the peninsula. The nested clade analysis provided evidence for past fragmentation events that may have been occurred during the Quaternary glaciations and had a major role in defining the genetic structure of the central-southern Italian beech populations. Only one haplotype apparently spread towards the north of Italy along the Apennine chain and reached the Italian slope of the western part of the Alps (Maritime Alps, Liguria). All haplotypes found along the Apennines remained trapped in the Italian peninsula. Southern and central Italy represent hotspots of haplotype diversity for Italian beech.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by O. Savolainen     

Chloroplast DNA diversity within and among populations of the allotetraploid Prunus spinosa L.

High chloroplast DNA (cpDNA) diversity was found within and among populations of Prunus spinosa sampled from seven European deciduous forests. A study of 12% of the total chloroplast genome detected 44 mutations, which were distributed over 24 haplotypes; four were common to two or more populations and the rest were unique haplotypes. The most-abundant and widely distributed haplotype was H2 (frequency = 41% approximately). Six of the seven populations were polymorphic. All of the six polymorphic populations had ”private” haplotypes (frequency < 5%) in addition to common haplotypes. The UPGMA dendrogram demonstrated a correlation between populations and their geographical locations. The total diversity was high (h_T= 0.824) and a major portion of it was within populations (h_s= 0.663). The level of population subdivision for unordered alleles was low (G_ST= 19.5%) and for ordered alleles was lower (N_ST= 13.6%). No phylogeographic structure could be demonstrated in the present geographical scale. High polymorphism in the cpDNA of P. spinosa has to be considered carefully when planning phylogenetic studies involving this species. Received: 20 September 1999 / Accepted: 10 November 1999     

Comparative phylogeography of bagrid catfishes in Taiwan

A phylogeographic analysis of two bagrid catfishes in Taiwan was conducted using sequence data from a portion of the mitochondrial DNA (mtDNA) control region. For Pseudobagrus brevianalis, which is most probably endemic to Taiwan, a total of eight haplotypes were detected in 189 specimens from nine river systems covering its entire distribution range, from northern to central western areas of the island. Obvious genetic differentiation was observed among its populations (average F _ST = 0.753); in particular, the northernmost Tamsui River population was fixed for a single endemic haplotype. Nested clade phylogeographic analysis (NCPA) indicated that the dispersal center of mtDNA was the area around the Touchien River and Holong River, north to the Miaoli Plateau, in northwestern Taiwan, suggesting both northward and southward dispersal in this species. There was no evidence for the validity of P. taiwanensis, the nominal species described from Taiwan, morphologically similar to P. brevianalis. We confirmed that P. adiposalis was distributed discontinuously in three river systems; analysis of 42 specimens from the rivers indicated a total of four haplotypes and population differentiation (average F _ST = 0.876). Fixation into a largely differentiated haplotype in the northernmost Tamsui River population was also found in this species, but different processes for this phylogeographic pattern were implied for the two species. Comparison with P. ussuriensis, a widespread continental species morphologically similar to P. adiposalis, suggested the possibility that P. adiposalis is a group of local populations of P. ussuriensis in Taiwan. Two migration routes vs. random fixation scenarios for the population structure of P. adiposalis are discussed with information on other fishes and the geological history of the island.     

Genetic variation of European grayling (Thymallus thymallus) populations in the Western Balkans

In order to elucidate genetic composition of European grayling (Thymallus thymallus) populations in the Western Balkans, the partial mitochondrial DNA (mtDNA) control region was sequenced and 12 microsatellite loci genotyped in 14 populations originating from tributaries of the Adriatic and Danube drainages. Eleven mtDNA haplotypes were found, one confined to the Adriatic clade, one to the Alpine group and the rest to the ‘Balkan’ grayling phylogenetic clade. Haplotypes from the Balkan clade were confined to the Danube drainage and constituted two groups: northern group with haplotypes found in the Slovenian part of the Danube drainage, and southern group, consisting from Bosnia–Herzegovina and Montenegro. Substantial genetic distance between northern and southern groups of haplotypes (0.75–1.8%) and well supported divisions within the northern group indicate very structured grayling population within the studied Danube basin that most probably did not evolve due to vicariance but rather as a consequence of multiple colonization waves that might have occurred during the Pleistocene. Furthermore, genetic distance of ~4% between Adriatic and Danube populations’ haplotypes, suggest that their separation occurred in mid-Pliocene. These findings imply a complex colonization pattern of the Western Balkans drainages. Microsatellite data also confirm high genetic diversity in Western Balkans populations of grayling (on average 7.5 alleles per microsatellite locus and H _exp 0.58). Limited stocking activities were detected based on microsatellites and mtDNA data. Regarding current knowledge of grayling phylogeography appropriate management strategies were proposed to preserve unique, autochthonous grayling populations in Western Balkan.     

Allozyme variations in Michelia formosana (Kanehira) Masamune(Magnoliaceae), and the inference of a glacial refugium in Taiwan

  Michelia formosana is distributed islandwide in Taiwan and also occurs in the Ryukyu Archipelago, Japan. Allozyme genetic variability in M. formosana (Magnoliaceae) was investigated using five polymorphic loci (Pgm-1, Idh-1, Mr-1, Skdh-1, and Ppo-3) from five enzyme systems. The average value of expected heterozygosity (H_e) describing within-population variation was 0.241. The overall F_is (0.0736) indicates a significant deficiency of heterozygotes at the population level. This positive value of F_is is mainly contributed from populations found at Pinglin, Chienshi, Wufeng, and the Ryukyus, all located at northern latitudes, and is also caused by locus Ppo-3. Among-population variation, F_st, accounted for 10.6% of the total heterozygosity and deviates significantly from zero. This populational differentiation agrees well with that of general tropical woody species outcrossed by animals. The population on Lanyu (Orchid Island) has some morphological differences from the plants native to Taiwan, but this was not reflected in the cluster analysis using the unweighted pair group method. Pgm-1d, however, is the diagnostic characteristic for the plants growing on Lanyu as it is entirely absent from all other populations of M. formosana. About 18% of the alleles of this study show clinal geographical variation and were found to be significantly related to latitudinal gradients throughout the species range. This, however, is not a linear relation but a curve with a peak form which is exactly the same as the relationship between expected heterozygosities of populations and latitude. This observation suggests that Nantou County, in central Taiwan could be a glacial refugium for genetic diversity of M. formosana. This inference is further supported by analysis of published studies on seven widely spread plant species in Taiwan. Location of this glacial refugium is probably related to the first emergence of the Central Mountain Ridge millions of years ago that naturally becamed the major window to receive genetic diversity from the Asian mainland. Received: 16 April 2000 / Accepted: 12 May 2000     

Cytoplasmic DNA variation and biogeography of Larix Mill. in Northeast Asia

Range‐wide variation in 54 populations of Dahurian larch (Larix gmelinii) and related taxa in Northeast Asia was assessed with four mitochondrial PCR‐RFLP and five chloroplast SSR markers. Eleven mitotypes and 115 chlorotypes were detected. The highest diversity was observed in the southern Russian Far East where hybrids of L. gmelinii, L. olgensis and L. kamtschatica are distributed. In contrast, only two mitotypes occurred in L. cajanderi and L. gmelinii. The Japanese larch (L. kaempferi) was found to be closely related to populations of L. kamtschatica inhabiting the Kuril Islands and South Sakhalin, populations from the northern part of Sakhalin being more closely related to continental species. In general, both mitochondrial (G_ST = 0.786; N_ST = 0.823) and chloroplast (G_ST = 0.144; R_ST = 0.432) markers showed a strong phylogeographical structure and evidence of isolation‐by‐distance. Yet both markers did not allow a clear delineation of species borders. In particular, and contrary to expectations, cpDNA was not significantly better than mtDNA at delineating species borders. This lack of concordance between morphological species and molecular markers could reflect extensive ancestral haplotype sharing and past and ongoing introgression. Finally the distribution of mtDNA and cpDNA variation suggests the presence of several refugia during Pleistocene glacial intervals. In particular, mtDNA and cpDNA reveal weak but visible differentiation between L. gmelinii and L. cajanderi, suggesting independent glacial histories of these species.     

Spatial pattern of chloroplast DNA variation of Cyclobalanopsis glauca in Taiwan and East Asia

This study examined the spatial pattern of chloroplast DNA (cpDNA) variation in Cyclobalanopsis glauca (Thunb. ex Murray) Oerst. (Fagaceae) in 140 trees from Taiwan (25 populations), Japan (three), Ryukyus (two), Hong Kong (one) and Mainland China (one). By sequencing three cpDNA intergenic spacer fragments using universal primers (trnT-trnL, trnV-trnM, including the trnV intron, and petG-trnP), we found a total of 1,980 bp and 15 polymorphic sites. Among them, 12 sites were caused by point mutation, and three resulted from insertion. This gives rise to a total of 13 cpDNA haplotypes. The level of differentiation among the populations studied is relatively high (GST = 0.612). Two ancestral haplotypes (A and B) are distributed widely in East Asia. Interestingly, all the derived cpDNA variations are found only in Taiwan but not in other areas. The Central Mountain Ridge (CMR) of Taiwan creates an unsurpassed barrier to the east-west gene flow of C. glauca. Among the populations on the west of CMR, only three separated populations, Yangmingshan, Wushe and Chinshuiying, have high haplotype diversity, each consisting of sister haplotypes all mutated from the same ancestral haplotype. Thus, they have probably originated from de novo mutation after the last glaciation. This inference agrees with the observation that no spatial autocorrelation existed on the west side. Two unrelated dominant lineages on the east of the CMR (haplotypes D and F) showed significant spatial genetic structure. Estimate of NST - GST was -0.090 and differed significantly from zero. Thus at the local scale, the phylogeographical component of the genetic structure is significant on the east of the CMR. Accompanied by published palynological records of the last glaciation, this study suggests the possibility that these two types were colonized northward from the southeastern part of Taiwan. 'Star-like' genealogy is characterized, with all the haplotypes coalescing rapidly and as a general outcome of population expansion (Page & Holmes 1998). A neutrality test also suggested a demographic expansion recovered from a bottleneck. We therefore inferred that the southeastern part of Taiwan might be a potential refugium for C. glauca.     

Hidden phylogeographic complexity in the Sierra Madre Oriental: the case of the Mexican tulip poppy Hunnemannia fumariifolia (Papaveraceae)

Aim A phylogeographic study of the endemic Mexican tulip poppy Hunnemannia fumariifolia (Papaveraceae) was conducted to determine: (1) the historical processes that influenced its geographical pattern of genetic variation; (2) whether isolation by distance was one of the main factors that caused genetic divergence in populations of this species; and (3) whether genetic flow still exists between populations from northern arid zones (Chihuahuan Desert and Sierra Madre Oriental) and those from southern arid zones (Tehuacán‐Cuicatlán Valley) – populations that are separated by the Transvolcanic Belt. Location Xerophytic vegetation in Mexico. Methods Chloroplast DNA (cpDNA) sequences of three regions, trnH‐psbA, rpl32‐trnL(UAG) and ndhF‐rpl32, were obtained for 85 individuals from 17 populations sampled in the field, covering the entire range of H. fumariifolia. The evolutionary history of these populations was investigated using a nested clade phylogeographic analysis and also by conducting various population genetic analyses. Results In total, 17 haplotypes were detected, 14 of which were found in the Sierra Madre Oriental. Differentiation among populations based on cpDNA variation (G_ST = 0.787, SE 0.0614) indicated population structure in H. fumariifolia, corroborated by a fixation index (F_ST) of 0.907. Results from analysis of molecular variance found that most of the total variation (90.71%, P < 0.001) was explained by differences among populations. Three regions were determined based on geological correspondence – the Chihuahuan Desert, Sierra Madre Oriental and Tehuacán‐Cuicatlán Valley – and the variation between them was significant (43.39%, P < 0.001). Results of a Mantel test showed a significant correlation between genetic and geographic distances (r = 0.511; P = 0.0001), suggesting a pattern of isolation by distance, which was corroborated by nested clade phylogeographic analysis. Mismatch distribution analysis indicated a sudden demographic expansion. Main conclusions Our study found that isolation by distance influenced genetic divergence in populations of H. fumariifolia. The finding that allopatric fragmentation influenced genetic divergence in populations in the Sierra Madre Oriental may be a reflection of the complex geology of the area. Our results suggest that the areas located in the north of the Sierra Madre Oriental acted as post‐glacial refugia for some populations.     

Comparative phylogeography of four component species of deciduous broad-leaved forests in Japan based on chloroplast DNA variation

A phylogeographic study of four tree species (Padus grayana, Euonymus oxyphyllus, Magnolia hypoleuca, and Carpinus laxiflora) growing in Japanese deciduous broad-leaved forests was conducted based on chloroplast DNA (cpDNA) variations. Using nucleotide sequences of 702–1,059 bp of intergenic spacers of cpDNA, 20, 27, eight, and eight haplotypes were detected among 251, 251, 226, and 262 individuals sampled from 67, 79, 75, and 71 populations of the above species, respectively. The geographical pattern of the cpDNA variations was highly structured in each species, and the following three regional populations were genetically highly differentiated among all four species: (1) the Sea of Japan-side area, (2) the Kanto region, and (3) southwestern Japan. Based on some interspecific similarities among the phylogeographic patterns, the following migration scenario of Japanese deciduous broad-leaved forests was postulated. During the last glacial maximum (LGM), the forests were separately distributed in six regions. After LGM, as the climate warmed, the forests in eastern Japan separately expanded from each of the refugia along the Sea of Japan-side or along the Pacific Ocean-side. In contrast, those in southwestern Japan retreated and moved to high altitudes from each of the continuous forests.     

Identification of glacial refugia in south-eastern North America by phylogeographical analyses of a forest understorey plant, Trillium cuneatum

Aim We examine several hypotheses emerging from biogeographical and fossil records regarding glacial refugia of a southern thermophilic plant species. Specifically, we investigated the glacial history and post‐glacial colonization of a forest understorey species, Trillium cuneatum. We focused on the following questions: (1) Did T. cuneatum survive the Last Glacial Maximum (LGM) in multiple refugia, and (if so) where were they located, and is the modern genetic structure congruent with the fossil record‐based reconstruction of refugia for mesic deciduous forests? (2) What are the post‐glacial colonization patterns in the present geographical range? Location South‐eastern North America. Methods We sampled 45 populations of T. cuneatum throughout its current range. We conducted phylogeographical analyses based on maternally inherited chloroplast DNA (cpDNA haplotypes) and used TCS software to reconstruct intraspecific phylogeny. Results We detected six cpDNA haplotypes, geographically highly structured into non‐overlapping areas. With one exception, none of the populations had mixed haplotype composition. TCS analysis resulted in two intraspecific cpDNA lineages, with one clade subdivided further by shallower diversification. Main conclusions Our investigation revealed that T. cuneatum survived the LGM in multiple refugia, belonging to two (western, eastern) genealogical lineages geographically structured across south‐eastern North America. The western clade is confined to the south‐western corner of T. cuneatum’s modern range along the Lower Mississippi Valley, where fossil records document a major refugium of mesic deciduous forest. For the eastern clade, modern patterns of cpDNA haplotype distribution suggest cryptic vicariance, in the form of forest contractions and subsequent expansions associated with Pleistocene glacial cycles, rather than simple southern survival and subsequent northward colonization. The north–south partitioning of cpDNA haplotypes was unexpected, suggesting that populations of this rather southern thermophilic species may have survived in more northern locations than initially expected based on LGM climate reconstruction, and that the Appalachian Mountains functioned as a barrier to the dispersal of propagules originating in more southern refugia. Furthermore, our results reveal south‐west to north‐east directionality in historical migration through the Valley and Ridge region of north‐west Georgia.     

Genetic variation in populations of the morphologically and ecologically variable fern <Emphasis Type="Italic">Stegnogramma pozoi</Emphasis> subsp. <Emphasis Type="Italic">mollissima</Emphasis> (Thelypteridaceae) in Japan

In Japanese Stegnogramma pozoi subsp. mollissima (Fisher ex Kunze) K. Iwats. there is the intrasubspecific variation among rbcL sequences. Northern and southern plants are genetically differentiated for maternally inherited cpDNA. In the present study we examined allozyme polymorphisms to test the hypothesis that northern and southern plants may be separate species. Based on allozyme data, the degree of gene flow among populations was estimated to be large. The artificial crossing experiments between cpDNA haplotypes also suggested that isolation has not developed among these cpDNA haplotypes. However, interpopulation genetic differentiation in cpDNA was observed even in the small area at the foot of Mt. Hakone, and the cpDNA haplotypes appear to have different habitat preferences. Received: May 1, 2001 / Accepted: September 30, 2001     

Genetic and morphological differentiation of the mangrove crab Perisesarma guttatum (Brachyura: Sesarmidae) along an East African latitudinal gradient

The genetic structure and morphometric differentiation of mangrove crab Perisesarma guttatum populations were examined among shelf connected locations along a latitudinal gradient on the East African coast. Over 2200 specimens were sampled from 23 mangrove sites for geometric morphometrics analysis. Population genetic analyses of mitochondrial cytochrome c oxidase subunit I (COI) DNA sequences were used to evaluate connectivity among populations. A total of 73 haplotypes were detected, and almost no haplotypes were found in common between two highly supported phylogeographic clades: southern Mozambique (Inhaca Island and Maputo Bay) and a northern clade that included north Mozambique, Tanzania and Kenya. These two clades were identified based on the species' populations pairwise genetic differentiation and geographical location. Φ_ST values were considerably high between the two clades, indicating the presence of significant population genetic structure between Kenya and South Mozambique. However, each clade was composed of genetically similar populations along the latitudinal gradient, and no significant population structure was found within each clade because the Φ_st values were not significant. The morphometric analysis corroborated the division into two clades (i.e. Inhaca Island/Maputo Bay and northern populations) and also detected less shape variation among populations that were few kilometres apart. The significant spatial genetic structuring between the southern and the northern populations of P. guttatum along the geographic gradient under study, combined with morphological differences, suggests that these populations may be considered as cryptic species. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 28–46.     

Intraspecific sequence variation of chloroplast DNA inPedicularis chamissonis Steven (Scrophulariaceae) and geographic structuring of the Japanese “Alpine” plants

In order to clarify evolutionary patterns and processes of intraspecific diversification ofPedicularis chamissonis Steven, we analyzed intraspecific variation of the nucleotide sequences of non-coding regions of chloroplast DNA: the intergenic spacers betweentrnT (UGU) andtrnL (UAA) 5′exon,trnL (UAA) 3′exon andtrnF (GAA), andatpB andrbcL. In 24 populations ofP. chamissonis, 33 nucleotide substitutions and 12 insertions/deletions were inferred, and their genetic distances ranged from 0.001 to 0.014. Seventeen distinct cpDNA haplotypes could be recognized and each haplotype was found to be geographically structured. Two major clades (the Northern and Southern clades) were revealed in phylogenetic analyses of cpDNA haplotypes. The haplotypes of the Northern clade had a wider distribution area in the populations of Mts. lide of central Honshu in Japan, northward to Unalaska Island in the Aleutians. Relationships among most haplotypes were unresolved polytomies. On the other hand, the haplotypes of the Southern clade occurred from the populations of Mt. Gassan southwards to Mt. Arakawa of central Honshu. Within this clade, three subclades were clearly recognized. From these results, we concluded that the haplotypes of the Northern and Southern clades inP. chamissonis might have traveled down to Japanese Archipelago from the north in not a single glacial period.     

Recent origin and adaptive diversification of <Emphasis Type="Italic">Ainsliaea</Emphasis> (Asteraceae) in the Ryukyu Islands: molecular phylogenetic inference using nuclear microsatellite markers

We investigated the phylogeny and phylogeography of four closely related Ainsliaea species (Asteraceae) on the continental Ryukyu Islands of eastern Asia, which consist of two flood-adapted “rheophyte” and two non-rheophyte (inland) species, based on 12 nuclear microsatellite loci. Phylogenetic analyses using 420 individuals from 26 populations showed that rheophytic A. linearis and A. oblonga are genetically distinct. Each species was clustered with the inland species that occur on the same islands, suggesting a different ancestry for the two rheophytes that evolved independently by local adaptation to flooded habitats. The results from the neighbor-joining clustering and principal coordinate analysis (PCoA) indicate that the southern populations of A. macroclinidioides are distinct lineages and ancestral to the northern populations as well as the other diverse species complex in the Ryukyus. These results suggest a pattern of colonization initially from the Asian mainland to the southern islands, followed by the northern islands via land bridges generated during the Quaternary glaciations. After isolation from southern populations, species radiation and regional differentiation within the northern clade occurred possibly via local adaptation and/or geographic isolation of the subdivided island bridge.