Biogeography and evolution of Asian Gesneriaceae based on updated taxonomy

Abstract Based on an updated taxonomy of Gesneriaceae, the biogeography and evolution of the Asian Gesneriaceae are outlined and discussed. Most of the Asian Gesneriaceae belongs to Didymocarpoideae, except Titanotrichum was recently moved into Gesnerioideae. Most basal taxa of the Asian Gesneriaceae are found in the Indian subcontinent and Indo-China Peninsula, suggesting Didymocarpoideae might originate in these regions. Four species diversification centers were recognized, i.e. Sino-Vietnam regions, Malay Peninsula, North Borneo and Northwest Yunnan (Hengduan Mountains). The first three regions are dominated by limestone landscapes, while the Northwest Yunnan is well-known for its numerous deep gorges and high mountains. The places with at least 25% species are neoendemics (newly evolved and narrowly endemic) which were determined as evolutionary hotspots, including Hengduan Mountains, boundary areas of Yunnan-Guizhou-Guangxi in Southwest China, North Borneo, Pahang and Terengganu in Malay Peninsula, and mountainous areas in North Thailand, North Sulawesi Island. Finally, the underlying mechanisms for biogeographical patterns and species diversification of the Asian Gesneriaceae are discussed.


Introduction
Gesneriaceae Rich. & Juss. ex. DC. is a middle-sized family, including about 150 genera and over 3400 species . Traditionally, the family was divided into two subfamilies, subfamily Cyrtandroideae Burnett (palaeotropical group, with superior ovary and two unequal cotyledons) and subfamily Gesnerioideae Burnett (neotropical group, with inferior ovary and two equal cotyledons) (Burtt 1998).
Based on recent molecular and morphological data, Weber et al. (2013) put forward a new classification system, consisting of three subfamilies: Sanangoideae Weber (monotypic genus, endemic to South America), Didymocarpoideae Arn. and Gesnerioideae Burnett. In this newest classification system, the monotype genus Titanotrichum Solereder in Asia (China and Japan) has been transferred to the subfamily Gesnerioideae, which was formerly treated as "New World Gesneriaceae" (Burtt 1998;Weber et al. 2013). However, Didymocarpoideae is still "Old World Gesneriaceae", consisting of 67 genera and more than 2300 species (Möller et al. 2017. Recently, many Asian Gesneriaceae taxa had experienced extensive revision for their systematic positions, such as Boea Commerson ex Lamarck , Microchirita (C.B.Clarke) Y.Z. Wang (Middleton 2018), Henckelia Spreng. (Middleton et al. 2013), Paraboea (C. B. Clarke) Ridley (Puglisi et al. 2016) and the enlarged genus Oreocharis Bentham Chen et al. 2014). Furthermore, several genera were newly established, i.e. Billolivia D.J. Middleton (Middleton et al. 2014), Chayamaritia D.J. Middleton , Middletonia D.J. Middleton (Puglisi et al. 2016;Puglisi and Middleton 2017), Rachunia D.J. Middleton . Such significant revisions of so many genera call for an updated study about biogeography and evolution of the Asian Gesneriaceae.
In this paper, we collected the species locality data (coordinates) from GBIF (Global Biodiversity Information Facility, https://www.gbif.org/) for all the species of the Asian Gesneraiceae. The species diversity and systematic positions of all genera were determined according to the newest literatures (e.g. Möller et al. 2016aMöller et al. , b, 2017Roalson and Roberts 2016;Puglisi and Middleton 2018). We used the software DIVA-GIS 7.5 to create a distribution map at 1° × 1° latitude/longitude grid resolution to reveal distribution patterns of species diversity and endemism. We also analyzed the distribution type of all the genera and identified evolutionary hotspots, i.e. the center of neoendemic species, which is determined when at least 25% of total species are locally endemic. Finally, we discussed the possible mechanisms, including both intrinsic and extrinsic factors, to explain the formation and maintenance of diversification and endemic centers of the Asian Gesneriaceae.

Distribution type
Based on Wu's (1979Wu's ( , 1991 criterions and Li's (1996) pioneer study on the geographical areal-types of the Asian Gesneriaceae, we identified the Asian Gesneriaceae as belonging to three areal types and 20 subtypes as below.

Geographical distribution patterns
Tropical and subtropical Asia are the distribution centers of the subfamily Didymocarpoideae, harbouring 85% genus and more than 90% species of Didymocarpoideae. Indo-China Peninsula and Southwest China (Figs 1, 2), which are dominated by limestone landscapes (Clements et al. 2006), are places notable for recording the highest species density ( Fig. 1 and Fig. 3). According to the updated phylogeny of the Asian Gesneriaceae (Möller et al. 2016b(Möller et al. , 2017Xu et al. 2017), most basal taxa of the Didymocarpoideae, like Rhynchotechum and Corallodiscus (Fig. 3), occur at India and Indo-China Peninsula and the nearby regions such as Sino-Vietnam region and Southwest Yunnan. This suggests that modern Didymocarpoideae probably originated in these regions.

Diversification and endemic centers
Our data recognized four species diversification centers (places with highest values of species density), i.e. Sino-Vietnam Region including boundary areas of Guizhou-Yunnan-Guangxi in Southwest China, Northwest Yunnan (Hengduan Mountains), Malay Peninsula and North Borneo (Fig. 3). In a study focusing on China's Gesneriaceae, Liu et al. (2017) found that richness of Gesneriaceae peaked in Southwest China, and Hengduan Mountains and boundary areas of Guizhou-Yunnan-Guangxi are the most significant hotspots of species diversity and endemism. Our results closely coincided with their findings.
Indo-China Peninsula turned out to be an extraordinary diversification center, harbouring several endemic genera Tribounia, Billolivia, Chayamaritia (Fig. 2). These genera are newly evolved and contain very few species (Fig. 4). The Indian subcontinent has the lowest value of species density, with only two endemic genera, i.e. Jerdonia and Championia (Sri Lanka). Boea was no longer widespread in tropic Asia and its endemic center appeared in Papua New Guinea and the Solomon Islands (Puglisi et al. 2016;Puglisi and Middleton 2018) (Fig. 2). Based on molecular data from Möller and Clark (2013) and Roalson and Roberts (2016), most locally endemic species such as in Aeschynanthus and Cyrtandra were newly evolved, i.e. neoendemics.  Black circles indicate diversification centers with highest species richness and the red grids are the evolutionary hotspots (at least 25% species are neoendemics). The species distribution information is obtained from http://www.gbif.org. The map was drawn using DIVA-GIS7.5.

Evolutionary hotspots
We determined places where at least 25% species are local endemics as 'evolutionary hotspots'. Six evolutionary hotspots were identified, i.e. Northwest Yunnan (Hengduan Mountains), boundary areas of Yunnan-Guizhou-Guangxi in Southwest China, mountains in North Thailand, Malay Peninsula, North Borneo, and North Sulawesi (Fig. 3). These evolutionary hotspots were distributed largely in the diversification centers ( Fig. 3), similar to findings of Liu et al. (2017) in which Hengduan Mountains and boundary areas of Yunnan-Guizhou-Guangxi were recognized as two hotspots of species richness of Chinese Gesneriaceae.
The geography of these six evolutionary hotspots was the most complex area in tectonic history, formed by the interaction of Indian, Eurasian, Australian and Pacific Plates (Hall and Spakman 2015). Therefore, the highly fragmented islands and limestone landscapes in Southeast Asia probably facilitated speciation of the Asian Gesneriaceae, similar pattern found in Begonia (Chung et al. 2014) and Alocasia (Nauheimer et al. 2012). Burtt (1998) proposed that Gesneriaceae is of southern hemisphere (Gondwana) origin, with the Gondwana broken down and dispersed all over the world. This hypothesis is based on Gesnerioideae spreading to South America via the Antarctic and Didymocarpoideae by migrating northwards, 'dropping' representatives in Africa and Madagascar and finally reaching the Eurasiatic continent and spreading from there to the Malay Archipelago and the Pacific. This hypothesis, however, faces difficulties both from the geological time scale and the molecular data. Perret et al. (2013) reconstructed the biogeography history and suggested an origin of this family in southern America during the late Cretaceous period. The Gondwana break-up, however, began at about 150 Ma (Hall and Spakman 2015). Woo et al. (2011) indicated that there were two independent long-distance dispersals or overland migrations from South America to Australasia via Antarctica, but how they entered Asia or Africa is still unclear. The molecular data from Möller et al. (2009) and Roalson and Roberts (2016) show that the most basal species were found on the Indian subcontinent, such as Jerdonia (mountains of SW India), Corallodiscus (Himalayas and China), Tetraphyllum, Leptoboea and Boeica (Himalayas and adjacent areas). Only Rhynchotechum, about 18 species, which is widespread from Himalayas to Malay Archipelago, and one other species, even reaches New Guinea. Furthermore, Boea was the only endemic genus at the east side of the Huxley's Line. Therefore, the Asian Gesneriaceae may have originated from the Indian subcontinent and/or Indo-China Peninsula, then dispersed to the east and the north and finally reached Southeast Asia and East Asia.

Origin and Evolution of the Asian Gesneriaceae
An up-to-date phylogeny indicated that Didymocarpoideae and Gesnerioideae probably separated at about 74 Ma (Roalson and Roberts 2016), when the Indian Plate had been separated from Gondwana (Hall and Spakman 2015). We propose two hypotheses for the origin of Didymocarpoideae. One is in India, whereby the ancestor of Didymocarpoideae dispersed via the south of South America and Antarctica to India, until about 45 Ma when the Indian Plate collided with the Eurasia Plate and rapid speciation occurred. This is in accordance with Burtt's (1998) "Out of India" hypothesis.
The new position of Titanotrichum in the Gesnerioideae (Wang et al. 2004) suggests a possible dispersal event from the New World to Asia. Perret et al. (2013) proposed that the ancestor of Titanotrichum might either disperse across Beringia from North America to East Asia or originate at East Asia. This genus is distinctive for small and numerous bulbils in the inflorescence, which evolved at about Miocene (Wang and Cronk 2003) and probably facilitated its long-distance dispersal.
The alternative hypothesis is that the Asian Gesneriaceae might follow the 'Malpighiaceae Route' via the 'North Atlantic land bridge' to Eurasia (Davis et al. 2002). However, currently very limited experimental studies focusing on this scenario have been carried out and we cannot speculate on details about this hypothesis. To test such a hypothesis, molecular biogeographic studies on a pantropical genus such as Epithema and Rhynchoglossum are needed to figure out their historical dispersal route(s) between tropical America, Africa and Asia.

Growth forms
According to Roalson and Roberts (2016), epiphytism and unifoliate growth are two important growth forms impacting on the diversification rate in Gesneriaceae, like Monophyllaea and a section of Streptocarpus Lindl. in the Old World. Epiphytes and non-epiphytes have the same speciation rate, but epiphytism has a much lower extinction rate (Roalson and Roberts 2016). This means that epiphytism per se is not the driving factor for speciation, it probably can lower the extinction rate or is associated with other traits that promoting speciation. For example, there is a strong correlation between the epiphytic habit and ornithocory (dispersal of seeds by birds) (Weber 2004). In orchids, epiphytism is also often correlated with CAM photosynthesis (Givnish et al. 2015), but in Gesneriaceae only the New World Codonanthe crassifolia was confirmed to have some CAM-like characteristics (Guralnick et al. 1986).
Unifoliate growth form strongly suggests that growth form positively influences speciation rate (Roalson and Roberts 2016). Previous work on Streptocarpus shows the evolution of growth forms, especially rosulate and unifoliate growth and they believed this was an adaptation for deep shade and unifoliate growth increasing diversification rate (Möller and Cronk 2001). Cyrtandra has, however, also undergone a significant increase in speciation rate, probably associated with other characteristics such as seeds' dispersal mode.

Specialized pollination adaptations
Normally, most Gesneriaceae species exhibiting zygomorphic flowers are thought to be more specialised in pollination adaptation, since it restricts pollinator behaviour and can therefore increase pollination efficiency (Gong and Huang 2009;Ling et al. 2017). Wang et al. (2010) and Ling et al. (2017) proposed that actinomorphy probably is a derived trait in the Asian Gesneriaceae, which is associated with shifts in pollination strategies, such as nectar-to pollen-rewards and/or from specialist to generalist pollinators.
Most Asian Gesneriaceae has the fused anthers, normally anthers are united by pairs, such as Loxostigma and Anna (Weber 2004). In some species, all the four anthers are fused or adnated, such as Beccarinda and Stauranthera (Weber 2004). Anther fusion can assemble the anthers to the same position and facilitate all the anthers touching the same locality of the pollinator's body, which can greatly enhance the precision of crosspollination (Ren 2008;Ren and Tang 2010). Wang (1990) indicated that anthers fusion with all stamens in the flower is late evolved than anther fusion in pairs and show a relatively higher level of pollination efficiency and consequently probably facilitate species diversifications.
There is a highly specialized pollination mechanism in the Asian Gesneriaceae, i.e. mirror-image flowers (Gao et al. 2006;Lu et al. 2019). Mirror-image flowers can be found in five genera in the Asian Gesneriaceae, i.e. Paraboea (Gao et al. 2006), Ornithoboea, Didymocarpus, Rhabdothamnopsis, andHenckelia (Lu et al. 2019). Mirrorimage flowers are a sexual polymorphism in which the style deflects either to the left or the right side of the floral axis (Jesson and Barrett 2002;Gao et al. 2006). Barrett (2002, 2003) pointed out that mirror-image flowers can increase the precision of cross-pollen transfer and may play an important role in pollination isolation and speciation. Normally, mirror-image flowers have reciprocal deflecting stamen(s) to the other side as compared with the deflecting style (reciprocal mirror-image flowers), which greatly increases pollen transfer efficiency (Jesson and Barrett 2003;Ren et al. 2013), but most mirror-image flowers in the Asian Gesneriaceae are non-reciprocal, without a deflecting stamen (Lu et al. 2019). Such unusual floral syndromes indicate unusual pollination adaptations in these species, but are awaiting further study.

Fruit adaptations to long-distance dispersal
In angiosperms, fruits significantly increase adaptive ability to withstand harsh environments and facilitate seed dispersal (Weber 2004). Fruit trait probably is a key trait for the high speciation rate and widespread range of both Aeschynanthus and Cyrtandra (Roalson and Roberts 2016). The hair-like appendages of seed in Aeschynanthus provide a favourable surface area to mass ratio (Denduangboripant et al. 2001), adapting to wind dispersal.
In Cyrtandra and Rhynchotechum, soft-fleshy (a true berry) fruits facilitate their colonisation throughout the Southeast Asia islands and nearby Pacific islands via bird dispersals (Cronk et al. 2005;Liu et al. 2017), and numerous islands in this area then promoted the speciation of the genus.

Extrinsic (environmental) factors
Compared to other regions, Asia is mostly dominated by the monsoon climate. There are three main types of summer monsoons in Asia, i.e. East Asia Monsoon, South Asia Monsoon and North-west Pacific Ocean Monsoon (Jiang et al. 2017;Kong et al. 2017). These three monsoons interact at Southwest China and Indo-China Peninsula and bring a large quantity of warm and wet air, thus providing a precondition for tropical plants to exist and facilitate the long-distance dispersal of the propagules. Monsoons do not only facilitate the northwards spread of tropical plants, but also aggravate the isolation of local habitat via the alternate dry and rainy seasons (Jiang et al. 2017), which might be related with the formation of diversification centers and evolutionary hotspots in Southwest China and Sino-Vietnam regions (Wang et al. 2011). More specifically, temperature changes since the Last Glacial Maximum had stronger effects on richness of rare species (Kong et al. 2017;Liu et al. 2017) while richness of common species was determined largely by current temperature seasonality such as monsoon climate .
Many studies had pointed out that microhabitat isolation caused by various landscapes such as limestone is the main factor for speciation in the Asian Gesneriaceae (Wang 1990;Ren 2015;Liu et al. 2017;Shui and Chen 2017). Especially for the three diversification and endemic centers, i.e. Sino-Vietnam Region, Malay Peninsula, North Borneo, the local landscapes are characterised by the various types of limestone landscapes (Clements et al. 2006;de Bruyn et al. 2014). Northern Borneo and Malaysia Peninsular were separated by the South China Sea, but they were connected during the glacial period and acted as a "land bridge" for plant dispersal across Southeast Asia (Hall and Spakman 2015). Frequent alternation of transformation between sea and continent intensified speciation in this area (de Bruyn et al. 2014). Southwest China has the largest continuous limestone areas in the world, which includes Guizhou, Yunnan, Guangxi provinces and the Sino-Vietnam region (Clements et al. 2006;Hou et al. 2010;Ren 2015;Kong et al. 2017). South Yunnan is not only rich in limestone landscapes, but also comprises a series of spectacular north-south trending ridges along three major rivers of Asia: the Salween, Mekong and Red River, which have formed many unique microhabitats and microclimates, such as dry and hot valleys in Yunnan (Jiang et al. 2017). These diverse landscapes, forming 'microhabitat islands', greatly facilitated plant speciation (Clements et al. 2006;Ren 2015). Clements et al. (2006) and Chung et al. (2014) also proposed that these kinds of limestone microhabitats were formed largely by the East Asian monsoon (Jiang et al. 2017).
Northwest Yunnan (Hengduan Mountains), located at the eastern fringe of the Tibetan Plateau, is widely recognised as a globally important biodiversity hotspot (Myers et al. 2000;Liu et al. 2017) and the cradle of new species with an extraordinarily high ratio of recently evolved endemic species (neoendemics) that resulted from the uplift of the Himalayas and surrounding mountains (López-Pujol et al. 2011). It is noteworthy that not only neoendemics of Gesneriaceae have been found here, but most of the basal taxa also appeared here (Möller et al. 2009;Möller and Clark 2013;Roalson and Roberts 2016). Such a distribution pattern may relate to the Asian Gesneriaceae migration route (Möller et al. 2009).
In conclusion, we have discussed the biogeographic and diversification patterns of the Asian Gesneriaceae, along with underlying mechanisms of the family's dispersal, adaptation and evolution. The family is still undergoing quick diversification and is awaiting further detailed studies not only about ecological adaptations but also evodevo examinations on relationships between micro-and macro-evolution. Molecular biogeographic studies on the typical pantropic taxa using updated techniques such as sequenced restriction-site associated DNA (Baird et al. 2008;Feng et al. 2017) are also suggested to explore the historical dispersal patterns and evolutionary diversification of the family from tropical America to Africa and Asia.