Impacts Of Spatio-temporal Patterns And Environmental Gradients On The Speciation And Evolution Of Isoetes (Isoetaceae) From China

Spatio-temporal patterns and environmental gradients have important impacts on speciation and evolution of plants. In traditional studies, however, scale effects in different space and time have been ignored, microevolution and macroevolution are apart discussed always, and interrelated hypothesis has been the scarcity of examples from nature. Isoetes (Isoetaceae) is a small, cosmopolitan genus of heterosporous lycopsids. The genus is considered to be the only remaining living representative of ancient taxa that were characterized by a strongly reduced plant body. It is an ancient group dating back to the Devonian and occupies a unique position in land plant evolution. Up to the present, origin and evolutional line, dispersal way and phylogeny of worldwide Isoetes are still uncertain, the mechanism of allopolyploid hasn't been revealed, and it has been unclear historical process and environmental factors how to impact speciation and evolution of Isoetes. Published studies on Isoetes in Mailand China are very limited. With special phylogenic position and geographical location, particular geological history, diversity distribution pattern and environmental gradients, Isoetes from China would provide a significant natural example for the study about impacts of Spatio-temporal patterns and environmental gradients on speciation and evolution.Based on spore morphology and chromosome number, four taxa of Isoetes from China are recognized: Isoetes hypsophila Hand.-Mazz., megaspores smooth, mean 358 μm in diameter, microspores with gyrus ornamentation, mean 22 μm in length, 2n =22; 1. yunguiensis Wang Q.F. & Taylor W.C., cristate-reticulate megaspores with mean 390μm in diameter, approximated smooth microspores with very fine granulate, mean 22μm in length, 2n =22; I. taiwanensis DoVel., tuberculatae megaspores with mean 312μm in diameter, echinatae microspores with mean 24um in length, 2n =22; I. sinensis Palmer, cristate megaspores with mean 409um in diameter, echinataemicrospores with mean 28μm in length, 2n=44. I. yunguiensis, which had previously been identified as I. japonica A. Br., is a new species completely different from the latter species. Comparing spore morphology and chromosome number among taxa, I. sinensis is regarded as allotetraploid with I. yunguiensis and I. taiwanensis as the putative parents. The hypothesis is supported by cloned sequence of the secondd intron a LEAFY homolog. Hybridization and allopolyploid are recognized as important way of evolution of Isoetes in China and East Asia, and the taxa from China play a significant role in the genus Isoetes from East Asia.Wild investigations show that dispersal of Isoetes is often accomplished via water current. According to analysis of nrDNA ITS sequence, Phylogeny of worldwide Isoetes including 9 populations of 4 taxa from China recognizes tree major well supported clades: an Indian clade {Isoetes coromadelina) (bootstrap =100%), an African-Europe-North American clade (/. nuttallii, I. orcuttii, I. abyssinica, I. velata, I. histrix) (bootstrap = 89%), and an Asian(China)-North American-Australian clade (/. hypsophila, I. yunguiensis, I. taiwanensis, I. sinensis, I. kirkii, I. brevicula, I. valida, I. echinospora, I. hawaiiensis) (bootstrap = 100%). On a background of large-scale global geological history, the analysis combining phylogeny, fossils and geographical distribution pattern suggests that the present pattern of Isoetes in global scale is the result of allopatric speciation derived from vicariance, dispersal and combination of both; East-South Siberia of Russia and Australia might be the two ancient centers of origin of the modern-day Isoetes and Annalepis is more probably ancestral to Isoetes; Chinese Isoetes might originate from Annalepis occurring in the Middle Triassic in the Yangtze River valley of South China and have very closely relationship with the Annalepis-type from East-South Siberia of Russia and Australia in Lower Triassic; Isoetes from China might be the key to explore the phylogenetic relationship of Isoetes in world scale; likewise, continental drift and Tethys evolvement have play an important role in dispersal and vicariance of Isoetes.The chromosome number was determined from eighty-one known populations of ten species of Isoetes in East Asia. The altitude was obtained in location of every population. T-test was used to evaluate the altitude difference between diploid and polyploid populations. Data of Japanese Isoetes from Takamiya et al. was used in the study. There are ten known species of Isoetes with four diploids and six polyploids in East Asia. The four diploid species (2w =22) are found at high altitudes (mean altitude = 2649.67+1396.16m, N=18) and isolated from each other. In contrast, the six polyploid species widely occur in low altitude regions (mean altitude = 182.62 + 181.44m, N=63) and are partially sympatric. Highly significant differences (p<0.001) were observed between the mean altitude of locations of populations of the diploid and polyploids. On the basis of large-scale pattern, combining geological history andgeographical change process in China and East Asia, it is suggested that the distribution of ancestral Annalepis shrunk gradually from east to west in the Yangtze River valley in response to the retreat of Tethys and the change of Chinese geomorphology from east to west during middle-late Triassic, subsequently, the ancestors of Isoetes could have reached the Qinghai-Tibet region before the beginning of the uplift of Qinghai-Tibet Plateau (Eocene of Lower Tertiary); these ancestral forms migrated from the high altitude to the low altitude areas during the generation of the stepped Chinese landscape via water flow from west to east, and result in the present distribution pattern of Isoetes in China and East Asia; the change of altitude might have played an important role in allopolyploid speciation and the pattern of distribution of the genus Isoetes in China and East Asia by exerting a considerable influence on dispersal and hybridization opportunities of Isoetes species, and the polyploidy speciations of Isoetes in East Asia might origin and develop from Holocene (Quaternary).20 parameters of water chemistry in the natural habitats of three species of Isoetes in mainland China were measured, using HANNA Pocket-sized pH Meter HI98107 (resolution = 0.1 pH, accuracy = ±0.1pH), HANNA Conductivity Meter Dist4/HI983004 (resolution = O.lmS. cm"1, accuracy = ±0.1mS. cm"1) and HANNA C200 Multiparameter Ion Specific Photometer (resolution of NO3-N , NO3", P, Mn and Mo = 0.1 mg. L"1, accuracy = ±0.1 mg. L"1; resolution of other factors = 0.01 mg. L"1, accuracy = ± 0.0 lmg. L"1). The character and variation of the chemical properties of the water were analyzed using mean, coefficient variability and difference significance tests (ANOVA and Kruskal-Wallis). Results showed a common character that there were bigger variations in average and coefficient variability of the 20 parameters. Average of Ni was overwhelm more than the other factors, and lower conductivity indicated there was litter pollution in the water. However, of the 20 factors assessed, except for pH (p<0.05), P (p<0.05), NO3-N (p<0.05), NO3"(p<0.05)and Al (pO.Ol), the rest displayed no significant difference among habitats of the three species. This indicated that most of the factors (75%) were relatively stable. pH had an identically positive correlation with NO3-N and NO3" (p<0.05), on the contrary, P had an identically negative correlation with NO3-N and NO3" (p<0.05). pH and P also had a negative correlation (p<0.05). Only Al had a positive correlation with P (p<0.05). An analysis, according to data from other research and this study, suggested the significant difference of chemical nature of the water among the three Isoetes species could be due to the difference of physiological character among them which might have a selective impact on their distribution.CCA (Canonical Correspondence Analysis) was used to estimate the impacts of environmental gradients on RAPD (Random Amplified Polymorphic DNA) genotypeof Isoetes, results show that 23 individuals from 9 populations of 3 taxa (/. hypsophila, I. yunguiensis, I. sinensis) in China are recognized as 5 type with significant difference consistent with the result of nrDNA ITS, and significant related to environmental gradients; four amongthe eight tested environmental factors including altitude (IC = 0.944) ^ P (IC = 0.883) >A1 (IC = 0.848) and longitude (IC = 0.820), have played important role in evolution of genotype, and altitude is the most major factor. It is suggested that evolution of genotype of Chinese Isoetes result from the combined effects of geographical isolation and ecological selection, and geographical isolation is the most important one; because of lack of gene flow, geographically isolated populations might generate evolution of genotype by ecological selection, however, if geographical isolation is absent, ecological selection to evolution of genotype would be blocked by gene flow and result in consistent genotype among population; likewise, phylogeny might respond to the history of geographical isolation and ecological selection, in other words, the history of speciation and evolution is closely related to the dynamic process of environmental evolvement.In a 4-year study the distribution and habitat characteristics of all four species from China (/. hypsophila, I. sinensis and /. yunguiensis on the mainland, and /. taiwanensis on Taiwan) were documented and their conservation status was evaluated using IUCN criteria. Our investigation allowed us to confirm 14 extant populations of four Isoetes species, including 10 populations recently discovered and 16 extinct populations of three species. All four species are facing a high risk of extinction, and should be categorized as Critically Endangered on the IUCN Red List. Habitat degradation and loss, water pollution and eutrophication, competitive exclusion, and human disturbance are the main factors causing the decline and extirpation of Isoetes. Only /. taiwanensis is protected in situ in Yangmingshan National Park on Taiwan. None of the species or habitats of Isoetes on Mainland China are presently protected, although all species have been designated by the government as State Key Protected Wild Plants. Our results suggest that conservation strategies should be implemented urgently, particularly on the Mainland.To sum up, spatio-temporal pattern and environmental gradient have an important role in speciation and evolution of Isoetes from China, and our understanding of the speciation and evolution would benefit from the studies combining dynamic space-time pattern and environmental gradients in different scale.