| Ceratopteris thalictroides (L.) Brongn., belongs to the genus Ceratopteris (Parkeriaceae), is a tetraploid, semi-aquatic, annual, homosporous fern with a wide geographic distribution in the tropical and subtropical regions of the world. In China, two species, C. pteridoides (Hook.) Hieron. (Diploid) and C. thalictroides, were recorded in Ceratopteris. Ceratopteris thalictroides grows mainly in agricultural fields, marshes, lakes, ditches, and ponds. Although C. thalictroides was widely distributed in China prior to the 1960s, it has since become severely vulnerable owing to the deterioration of its primary habitats as a result of human activity and the number of populations has declined rapidly. During our field investigations, only 24 small extant populations of C. thalictroides were found. The species is now considered to be endangered in China and is listed as the second category of key protected wild plants. Thereis now an urgent need to study the conservation biology of this endangered species. Ceratopteris thalictroides is highly plastic in morphology with a number of forms. Three cryptic species were revealed in the previous studies based on cell biology observations, artificial crossing, allozymes markers and the chloroplast DNA sequences, referred to as the north type, the south type, and the third type. The Chinese C. thalictroides and Japanese C. thalictroides plants are close distributed in east Asia. It is possible that there exist some cryptic species in Chinese C. thalictroides. Although the specimen sampling in the previous cryptic species diversity study covered a large geographical region, sampling in China was confined to Taiwan and much of the distribution range of C. thalictroides in China was not included in the sampling of the previous studies, the taxonomic complexity of C. thalictroides in China remained uncertain. In addition, previous population genetic studies sampled only 13 populations of C. thalictroides in China, which has provide valuable information on the conservation genetics of this endangered species, however, what is the level and pattern of the genetic variation in the newly discovered populations? This question is still remain unanswered. It is necessary to answer all these questions before making a sound protect strategy for the conservation of C. thalictroides in China.Molecular phylogeographic studies often reveal evidence of cryptic variation, and in many cases lead to the discovery of new species. A phylogeographic approach can reveal the nature of evolutionary diversification and biogeographical segregation. In order to clarify how many cryptic species are present within C. thalictroides in China, infer the phylogeographic patterns of the cryptic species, and reveal the genetic structure of C. thalictroides, in this study we first analyzed the genetic structure of the cpDNA non-coding regions identifying haplotypes and their distribution throughout the entire range of C. thalictroides in China, then we use the co-dominant SSR markers developed in this study to study the genotype diversity of this endangered species. These efforts may facilitate in situ and ex situ conservation management of this endangered species in China by allowing the full range of genetic variability to be protected. The main results and conclusions of this study are listed here below:(1) We examined four chloroplast DNA (cpDNA) non-coding regions and compared sequence variation within C, thalictroides complex. Sequence data were obtained from 143 individuals in 24 populations throughout the natural distribution of the species in China. Nineteen haplotypes were identified. Molecular systematic and phylogeographical analyses revealed two genetically distinct clusters of cpDNA haplotypes in China. One cluster included haplotypes associated with the north type, and another with the south type cryptic species. The NST value was significantly higher than the GST value (NST=0.768> GST= 0.434, P<0.05), indicating the presence of a significant phylogeographical structure of C. thalictroides in China. The results of AMOVA analysis showed a significant inter-group differentiation (FST= 0.918; P< 0.001). Analyses based on different, but complementary methods suggest that in China, C. thalictroides contains only two of the cryptic species (the north and south types). Two haplotypes, H8 and H17, of the interior node in the minimum-spanning network (MSN) of cpDNA haplotypes are widespread. The origin of the widespread haplotypes in China may have resulted from long-distance dispersal to China. We propose that Indonesia is likely to be the center of origin and distribution for the cryptic species in C. thalictroides. Indonesia is located in the Indian subcontinent and is a neighboring China. It is highly possible that the cryptic species in Indonesia dispersed to China. For the north type cryptic species, the Island of Taiwan seem like probable source areas for the initial to colonization of mainland China with the subsequent dispersal to the tropical and subtropical regions of China. For the south type cryptic species, Island of Hainan seems the likely place of colonization prior to immigration to the HP population in Guangxi province.(2) We used RAD tag sequencing technique to develop microsatellite markers. An individual of C. thalictroides from Wuhan Botanical Garden was used as the source of DNA in this work. The RAD library sequenced at a Illumina HiSeq 2000 Platform, generating 0.45 million DNA reads. A total of 650 SSR loci were identified using MIcroSAtellite identification tool (MISA) from the resources and 285 primers were successfully designed using Primer3. The random selected 60 of the designed primers were initially screened using total DNA isolated from dried leaves of six C. thalictroides individuals. Based on the initial screening results, eight primer pairs were selected and used to genotype 26 individuals from two populations located in Baise of Guangxi Provinceand Yingde of Guangdong Province, respectively. The number of alleles per locus ranged from 4 to 8. The expected heterozygosity (HE) values and the Shannon-Wiener index (H') ranged from 0.438 to 0.814 and 0.676 to 1.828, respectively. Because these eight microsatellite markers exhibit high degrees of genetic variation, they should be useful tools for studying the adaptive genetic variation and sustainable conservation of C. thalictroides.(3) We genotyped a total of 229 individuals from 24 populations of C. thalictroides in China using six newly developed SSR markers (Cerl-6). The C. thalictroides is a tetraploid fern, in this study we used two methods to generate the SSR genotype data and used two software (ATETRA and GenAIEx) to study the genetic diversity and structure of 24 populations of C. thalictroides. ATETRA software takes into account all possible combinations of allele copy numbers in populations with partial heterozygotes, for every locus and for every population individually. For the GenAIEx software, we treated the codominant microsatellite alleles as loci, creating binary genotypes ("presence" and "empty") and subsequently analyzed them as dominant data. Both of the software (e.g., the results from NJ tree, AMOVA, PCoA, genetic differentiation, and genetic distance evaluations) revealed a similar pattern of genetic differentiation between populations of SY, YC and HP, and the rest studied populations, supporting the finding that there exists two cryptic species in Chinese C. thalictroides plants. However, the two software revealed a different within-population genetic diversity, for example, the ATETRA generated a high level of allelic diversity, but the GenAIEx generated a low level of percentage of polymorphic loci (PPL). We proposed that it might be due to different statistic methods were used in allele counting and the method that treated the codominant microsatellite alleles as loci might have underestimated the genetic diversity. However, it is need to further compare these two methods. Although it is still inconsistent in the within-population genetic diversity, for each cryptic species, a low level of among-population genetic differentiation was revealed by both methods. No significant genetic differentiation between populations within groups indicated the regular occurrence of high gene flow. However, we do not propose efficient ongoing gene flow between extant populations of each of the cryptic species. Considering the fragmentation of the present-day habitats, we suggest that high gene flow represents shared ancestral polymorphisms within populations. The clonal habit of this species may have contributed to retention of genetic diversity in the remnant populations of C. thalictroides by " fixing " some of the genetic variation.Our results based on four non-coding spacers of cpDNA and six SSR molecular markers show that there exist two genetically distinct cryptic species in C. thalictroides in China. These therefore need to be treated as separate management units when establishing a conservation program. For each cryptic species of C. thalictroides in China, damage to the habitats is the prevalent causative factor for the decline in populations and number of individuals, conservation strategies should aim at protecting more habitats. |
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