Towards an Understanding of the Distribution of Ilex L. (Aquifoliaceae) on a World-Wide Scale

BS 55 501 Towards an understanding of the distribution of Ilex L. (Aquifoliaceae) on a World-wide scale

PIERRE-ANDRÉ LOIZEAU, GABRIELLE BARRIERA, JEAN-FRANÇOIS MANEN AND OLIVIER BROENNIMANN

LOIZEAU, P.-A., BARRIERA, G., MANEN, J.-F. & BROENNIMANN, O. 2005. Towards an understanding of the distribution of Ilex L. (Aquifoliaceae) on a World-wide scale. Biol. Skr. 55: 501-520. ISSN 0366- 3612. ISBN 87-7304-304-4.

Almost 600 species of Ilex L. (Aquifoliaceae) are now recognized and most of them occur in tropical America and eastern Asia. Only c 30 species are known from North America, four species are found in Europe, a few on Pacific islands, one in northeastern Australia and one in sub-Saharan Africa. Fossil data shows that this genus previously had a much wider distribution. Fossils have been found in Alaska and Iceland, western North America and southern South America, Siberia, New Zealand and southern Australia. Floral morphology of Ilex species is very uniform at an inter- specific level, whereas leaf morphology often shows great variability at an intra-specific level resulting in difficulties of discriminating between the different species. We compared recon- structed phylogenies based on chloroplast and nuclear DNA sequences, and morphological characters from 47 Ilex species. The plastid phylogeny is strongly correlated with the geographic distribution of extant species. However the plastid and nuclear phylogenies are not congruent. This may be due to frequent inter-lineage hybridization events, a process which could explain the high complexity of this family (i.e. at morphological, genetic, geographical distribution levels). We did not obtain a resolved phylogeny based on morphological characters. A world-wide distribution modelling map has been computed by an Ecological Niche Factor Analysis (ENFA) based on 826 Ilex occurrences in tropical America and 12 GIS layers describing the environment. The map of the potential distribution obtained is consistent with the distribution of the genus at present. However the model has less predictive probability for the northern hemisphere. Data of pollen fossil records for all Ilex over the world were mapped at world scale at various geological periods. Analyses of extant and past distributions of the genus, and phylogenetic results integrated with climatic patterns have been used to tentatively explain the current distribution pattern of Ilex. Some hypothesis assuming Bering and North Atlantic land bridge connections, Tertiary relict flo- ras, long distance dispersal or morphological stasis are congruent with the current disjunct distribution of the family.

Pierre-André Loizeau, Conservatoire et Jardin botaniques, Chambésy/Genève, Switzerland. E-mail: pierre- [email protected]

Gabrielle Barriera, Conservatoire et Jardin botaniques, Chambésy/Genève, Switzerland. E-mail: [email protected]

Jean-François Manen, Conservatoire et Jardin botaniques, Chambésy/Genève, Switzerland. E-mail: [email protected]

Olivier Broennimann, Université de Lausanne, Institut d’écologie et de géobotanique, Dorigny/Lausanne, Switzerland. E-mail: [email protected] 502 BS 55

Introduction deciduous or evergreen, the size and shape of leaves, complexity of the cymose inflores- Diversity and distribution cences, and the number of floral parts. The fol- Aquifoliaceae, the holly family, was once repre- lowing large-scale anatomical studies empha- sented by four genera: Ilex L., Nemopanthus sise some of the difficulties encountered when Raf., Phelline Labill. and Sphenostemon Baill. discriminating between different species (Cronquist 1981), but today it includes only within the genus. Baas (1973) published an the genus Ilex (Judd et al. 1999; Powell et al. extensive treatment on the wood anatomy of 2000; Loizeau et al. in press), which is com- Ilex. He concluded that Ilex species were impos- posed of almost 600 species. Although cos- sible to separate using wood anatomy alone. mopolitan, it is very unevenly distributed in He made the same conclusion based on a study terms of the number of species occurring in of leaf anatomy (Baas 1975). Lobreau-Callen each continent. Ilex occurs mostly in the trop- (1975) studied the pollen of more than half ics but extends into temperate regions with the species included in Loesener’s works oceanic climates up to 63°N (America, Eura- (1901, 1908) and concluded that Ilex species sia) and down to 35°S (America, Africa). There could not be separated based on pollen charac- are c 300 species in tropical America (Loizeau teristics. Loizeau and Spichiger (1992) pro- 1994), c 250 species in south-east Asia, c 30 posed a classification based on the structures species from North America, four species in of the inflorescence in Ilex. They included an Europe (including the endemic species of the evolutionary character for the first time into Canary Islands, Madeira and Azores), a few the classification of Ilex. species in Pacific islands, one in northeast Aus- tralia and one in sub-Saharan Africa. Tropical Ancient and extant pollen America and south-east Asia are the important The pollen of Ilex is very characteristic (Martin centres of species diversity for this genus. 1977) and no other known pollen type could Species of Ilex are found from lowland to mon- be confused with it (Fig. 1). The closest pollen tane forest (disturbed or primary) up to 4000 grain morphology to Ilex is that of Coprosma meters elevation in the Andes and in the nertera F. Muell. (Rubiaceae), which has other Himalayan region. The family is usually found in humid habitats (Martin 1977) and is totally absent from very dry areas.

Systematics A world-wide treatment of the genus was done by Loesener (1901, 1908, 1942). Floral morphology of Ilex species is very uniform at a inter-specific level whereas, leaf morphology often shows great variability at an intra-specific level, resulting in difficulties in discriminating between different species. Ilex is composed of dioiceous shrubs or trees with simple and alter- nate leaves, and small, usually 4-5-merous flow- ers gathered in cymose inflorescences. Species are mostly recognized on whether they are Fig. 1. Ilex teratopis pollen grain (photo J.Wuest). BS 55 503 features that readily distinguish it. The other Asian Ilex of the Bonin and Ryukyu Islands. taxa with a pollen ornamentation resembling These two studies both demonstrated natural that of Ilex can easily be separated using other inter-specific hybridization in Ilex. In horticul- pollen morphological characters. Ilex represen- ture, inter-specific hybridization is achieved tatives produce low amounts of pollen, they are relatively easily between different species of the insect-pollinated and very little pollen is found genus (Galle 1997). in the atmosphere (Hyde 1961; Clot pers. Finally, a relative test of the rate of comm.). Moreover they have a relatively low nucleotide substitution made by Cuénoud et al. pollen dispersal capacity (Behling et al. 1997). (2000) gave the age of the common ancestor of The oldest findings of pollen attributed to Ilex the extant species as 50 million years old. This originate from the Turonian or earliest upper age is far from the 90 mya indicated by the fos- Cretaceous in Australia, 90 million years ago sil records. According to these authors this dif- (mya) (Martin 1977). But the genus was appar- ference could be explained by extinction of ently already cosmopolitan by the late Cre- the basal branches of extant species, which taceaous, since pollen grains of Ilex dated at 70- then do not represent the entire lineage. 85 mya have been found in Africa, western Moreover they consider the Eocene (54-36 North America, and South America (Lobreau- mya) as an important era for diversification in Callen 1975; Martin 1977; Muller 1981). How- this genus. ever, few data (pictures) of these very old fossil Two questions that this paper attempts to pollens of Ilex have been published. clarify are: (1) Can contradictions between the past and present distribution of Aquifoliaceae Molecular analyses be explained using a comparative analysis of The phylogenetic history of the genus based fossils and the current distribution of Ilex, that on DNA analyses has been studied by Cuénoud also integrates the different theories involving (1998), Cuénoud et al. (2000), Setoguchi and the earth’s history (continental drift, migra- Watanabe (2000), and Manen et al. (2002). tory pathways, climate fluctuation) from 90 The chloroplast atpB-rbcL spacer has been mya? (2) Can a phylogeny of Ilex, based on sequenced for 116 taxa represented in most morphological characters, improve the under- parts of the world (Cuénoud et al. 2000). The standing of the different migration patterns in plastid phylogeny showed four major clades the genus? that were poorly supported and lacked any resolved hierarchy between them (i.e. an American, two Asian/N. America, and a Materials and methods Eurasian clade). Manen et al. (2002) compared Morphological analyses a three-gene plastid phylogeny with a two-gene For the 47 Ilex species from all over the world, nuclear phylogeny based on 47 species selected for which plastid and nuclear phylogeny was among the taxa studied by Cuénoud et al. available (Manen et al. 2002), morphological (2000). They observed an incongruence descriptions including vegetative (leaves, ram- between the two phylogenies, which they con- ets, etc.) and sexual (inflorescence, flower and sidered due to the probable expression of a fruit) characters were made. Descriptions were strong inter-specific and interlineage based on herbarium specimens from BM, BR, hybridization, making phylogenetic studies COL, F, G, K, MBM, NY, P, S, US, and W. When very complex. The same conclusion was made necessary, the descriptions were supplemented by Setoguchi and Watanabe (2000) based on with information from literature, mostly Loe- 504 BS 55 sener (1901) and Galle (1997) (Appendix 1). the distribution of ecological values for the Within the framework of our research on whole study area. Species distribution based on Neotropical Aquifoliaceae, 126 morphological the ENFA factors is then used to compute a characters were chosen to infer phylogenetic habitat-suitability map (Hirzel et al. 2002). relationships among the 47 species included in Model validation is achieved in Biomapper the present study. through a jack-knife cross-validation process A matrix of characters was built up for the 47 (Fielding & Bell 1997) with presence points representative species (available at the address partitioned in ten subsets of equal sizes. of the first author) and was used to generate The study area comprises the world-wide morphological trees with PAUP 4.0b3a (Swof- land area, excluding Antarctica. All analyses ford 2000). The trees were arbitrarily rooted have been performed within a raster-map data with Ilex canariensis Poir., which is claimed to be structure based on the latitude/longitude the most basal taxon (Manen et al. 2002), to coordinate system with a 0.5 decimal degree compare their topology with the molecular resolution. The Ilex presence dataset origi- phylogenetic trees. nated from a tropical American Aquifoliaceae The plastid and nuclear trees (Manen et al. database (Conservatoire et Jardin botaniques, 2002) were used to analyse the character Geneva) based on herbarium specimens. The matrix. Consistency indices (CI) of each char- study included 3506 individuals and each was acter were tabulated using MacClade (Maddi- described by its geographic coordinates. Only son & Maddison 1992). one location occurring in the same 0.5 x 0.5 degree grid cell was kept, the final sample size Distribution modelling was thus reduced to 826 occurrences. The sec- In the classical approach, distribution model- ond type of data required for the ENFA is a set ling (e.g., GLMs, McCullagh & Nelder 1989; of quantitative raster maps describing the envi- GAMs, Hastie & Tibshirani 1987) requires both ronment. A mean monthly climate dataset presence and absence data. The available Ilex (CRU global climate dataset, New et al. 1999) data do not allow us to use this kind of model was used to create annual climatic predictors. because, as is often the case with herbarium Monthly data consisted of data to 0.5 degree material, the absence of herbarium collections latitude/longitude resulting from an interpola- from a given place may indicate that taxa are tion from worldwide meteorological stations. absent or that there are no collections from Annual data have been produced through GIS that region. To overcome this problem of calculation, resulting in 12 GIS layers. absence data, several modelling techniques which incorporate presence data only have Pollen fossil distribution been developed in recent years (e.g., BIO- A database of the pollen fossil records for Ilex CLIM, Austin et al. 1994; GARP, Peters & from all over the world has been compiled Thackway 1998; ENFA, Hirzel et al. 2002). In from published sources cited by Martin (1977), this paper, the distribution modelling map has Muller (1981) or directly from Wijninga and been computed by ENFA (Ecological Niche Kuhry (1993). The data were mapped at world Factor Analysis) using the software Biomapper scale (Scotese 2003) at various geological peri- (Hirzel et al. 2002). The distribution modelling ods with for each period the corresponding map is then called habitat-suitability map. position of the continents. The object was to ENFA allows us to compare the distribution analyse the distribution of fossil records in an of ecological values for a presence data set to attempt to determine the origin and phytogeo- BS 55 505

Fig. 2. Strict consensus of 22 most parsimonious trees (820 steps, CI = 0.20, RI = 0.42) obtained from the matrix of characters using the heuristic search (TBR, 100 random taxon additions) of PAUP. Bootstrap values (if > 50) are indicated below the branches. Abbrevia- tions of the geographic distribution are alphabetically: Afr = Africa, And = Andes, Bra = Brazil, Cam = Central America, including South Mexico, Can = Canary Islands, Car = Caribbean Islands, Eas = East Asia, Eur = Europe, Gui = Guiana, Haw Tah = Hawaii/Tahiti, Mac = Macaronesia, Nam = North America, Sam = South America, Sea = South-East Asia. 506 BS 55

Fig. 3. Habitat-suitability map for Ilex, as computed by Ecological-Niche Factor Analysis (ENFA). Ilex occurrences from which ENFA was computed are illustrated with black dots. The habitat suitability values are represented as follows: white areas 0-20%, light-gray areas 20-40%, gray areas 40-60%, dark-gray areas 60-80% and black areas 80-100%. Hatched areas correspond to the extant distribution of the genus. The model accuracy is given by the strong values of the mean (65,53) and the standard deviation (28,04) of values predicted by the model for the occurrences.

graphic relationships of modern Ilex distribu- Distribution modelling map tion. The macrofossils were not retained The genus Ilex shows a global marginality value because they are not considered a reliable data of 0.939 and a global tolerance value of 0.292, source unless they are identified through indicating as expected that its habitat differs autapomorphies shared with extant species from the world average conditions and that its (Hill 2001). ecological niche is relatively restricted. The first four ENFA factors account for 92% of the variance. The marginality factor alone explains Results 72% of the variance. Its coefficients show that Morphological analyses Ilex distribution is essentially linked to indices The morphological tree (Fig. 2) is incongruent of precipitation (0.49 for the year, and 0.41 for with either the plastid or nuclear tree. The both the driest and wettest months) and to a Consistency Index (CI) of each character was lesser extent temperature during the coldest calculated on the plastid and nuclear trees to month (0.33) and number of frost days determine any correlation between morpho- (- 0.31). The suitability map is based on all the logical characters and molecular trees. No cor- ENFA factors. The jack-knife cross-validation relation was found with any of the molecular shows that predicted suitability exceeds 0.5 in trees (results not shown). 69.6% of the validation cells, which proves that the model is well supported. BS 55 507

The map of potential distribution obtained evolution and a very broad distribution, sug- (Fig. 3) is consistent with the distribution of gests that speciation within the genus is very the genus at present. However, due to all the slow (but see Burnham & Graham 1999). geographical occurrences being from Central and South America, the model has a lesser pre- Differentiation of extant Ilex species dictive probability for the northern hemi- The very complex history of the genus Ilex is re- sphere (sampling bias). Nevertheless, the flected in our incapacity to produce a relevant model is sufficiently relevant to allow an inter- cladogram of morphological characters, which pretation of the differences between potential is also underlined by the incongruence ob- and observed distribution compared to the served between the plastid and nuclear phylo- paleo-environmental variations. genies. The great potential of inter-specific hybridization of Ilex (Galle 1997), associated with Pollen fossil distribution multiple migration pathways defined by Cué- Pollen fossil records have been plotted from noud et al. (2000) could explain our incapabili- the Mid-Cretaceous through to the Miocene. ty to achieve a hierarchical arrangement of the Figure 4 shows the geographical distribution of species. It thus appears to be impossible to use Ilex fossil records at different geological peri- the evolutionary tendencies of the genus to ods considering continental drift. analyse the cladograms on a hierarchical basis. Several studies of wood anatomy (Baas 1973), leaves (Baas 1975), and pollen (Lobreau-Callen Discussion 1975; Martin 1977), show that it is impossible to Past species diversity distinguish the different species on the basis of Palynological data indicate only the presence these characters. Furthermore, these authors or absence of the genus in a place at a given observed great homogeneity within the extant time, but give no information on the specific species throughout the world. diversity of Ilex in the past as the species cannot At a morphological level, the extant species be distinguished based on pollen morphology are often defined by continuous variation of only. In terms of distribution of the occur- forms and/or dimensions. Within the study of rences in space and time, such records do not South American species, the difficulty over sep- give any information on the migratory path- arating the different species has not been ways or the centres of diversity. resolved. Often, the samples can be classified As outlined by Cuénoud et al. (2000), pollen on a gradient which passes imperceptibly from fossil records taken from the literature seem to one species to the other (e.g., Ilex kunthiana indicate that the Ilex lineage was already cos- Triana, I. myricoides Kunth, I. ovalis (Ruiz & mopolitan long before the end of the Creta- Pav.) Loes., I. rupicola Kunth, I. scopulorum ceous. Cuénoud et al. (2000) and Manen et al. Kunth, I. uniflora Benth.). A biometric study in (2002) arrived at the conclusion that the origin progress on Ilex laurina Kunth, I. yurumanguinis of Ilex is c 50 mya, on the basis of phylogenetic Cuatrec. and I. maxima W. J. Hahn confirms study of extant taxa. They do not dispute the the existence of this gradient without the possi- possibility that older ancestors existed, but bility of morphologically separating them with those would belong to currently extinct the exception of the typical specimens of each branches. taxon. A certain number of samples could be The high homogeneity of extant species of interpreted as being hybrids between I. laurina the genus, in spite of at least 50 million years of and I. yurumanguinis. 508 BS 55

Fig. 4 (A-B). Ilex fossil distributions at different geological periods. Position of landmasses according to Scotese 2003. Posi- tion of pollen according to Martin (1977), Muller (1981) and Wijninga and Kuhry (1993). A) Miocene. B) Oligocene. The periods of time corresponding at the position of the continents are: A) 10 mya. B) 30 mya. BS 55 509

Fig. 4 (C-D). Ilex fossil distributions at different geological periods. Position of landmasses according to Scotese 2003. Posi- tion of pollen according to Martin (1977), Muller (1981) and Wijninga and Kuhry (1993). C) Eocene. D) Paleocene. The periods of time corresponding at the position of the continents are: C) 40 mya. D) 60 mya. 510 BS 55

Fig. 4 (E-F). Ilex fossil distributions at different geological periods. Position of landmasses according to Scotese 2003. Posi- tion of pollen according to Martin (1977), Muller (1981) and Wijninga and Kuhry (1993). E) Late Cretaceous. F) Mid-Cre- taceous. The periods of time corresponding at the position of the continents are: E) 70 mya. F) 90 mya. BS 55 511

These facts suggest that the number of Bering land bridge probably allowed the pas- extant species of Ilex could be greatly reduced sage of deciduous taxa of Ilex for a longer after taxonomic study. period of time than for the evergreen ones, because of the capacity of deciduous species to Extant distribution survive in a colder climate by losing their A disjunct distribution pattern is observed leaves. The deciduous species seem to be the between eastern Asia and eastern North Amer- most recently evolved as an adaptation to the ica, as there are no Ilex species along the west- cool periods that appeared during the late Ter- ern North American coast. This disjunct distri- tiary. But no information is available that bution pattern has been known for many years would allow us to know when deciduous taxa and for numerous taxa (Graham 1972; Raven appeared, or on which continent, or if they 1972; Raven & Axelrod 1974; Boufford & appeared separately on each continent. Plastid Spongberg 1983). data suggest it happened twice, both in North This general phytogeographical pattern can American/Asia disjunctions. be directly explained by the broad distribution Wen (1999) showed that most intercontinen- of elements of northern hemisphere forests tal species pairs are not sister species, which during the mid-Tertiary and subsequent could mean that species pairs found in both colonisations in north-western America and continents do not necessarily have a direct phy- western Europe in response to climatic cooling logenetic link. Three reasons could be invoked at the end of the Tertiary and during the Qua- to explain this morphological similarity: (1) ternary (Wen 1999). The Bering and northern the pairs of species have been separated for a Atlantic land bridges probably contributed to very long time and did not evolve much before the floristic exchanges between southeastern or after their separation (Wen 1999), and a Asia and North America. Tiffney (1985) pro- morphological “dormancy” theory (stasis) is posed five possible principal periods for these suggested to explain this, (2) the morphologi- exchanges: Pre-Tertiary, beginning of Eocene, cally similar species are the product of similar end of Eocene-Oligocene, Miocene, and the evolution in distant, but equivalent environ- end of the Tertiary-Quaternary. ments. Qiu et al. (1995) believe that the similar In eastern Asia, the majority of genera with a type of habitat can exert a comparable pres- disjunct distribution between southeastern sure, which influences in a convergent way the Asia and eastern North America are present in morphological adaptation, and 3) pairs of the Sino-Japanese floristic area (Boufford species are not so morphologically similar as 1998). This area extends from western Yunnan previously suggested. and Sichuan, through eastern and southern These observations could explain why the China, to eastern Korea and Japan (Wen plastidic, nuclear and morphological phyloge- 1999). The Sino-Japanese area is additionally nies of the Ilex are not congruent. rich in endemics (Wen 1999). The specific relations between North Amer- Ilex seems to follow this pattern of distribu- ica and south-eastern and eastern Asia should tion for the eastern and south-eastern Asian be studied more deeply to see if sister species and North American species. In the former are present on these two continents. Some of area great species diversity is found whereas in the deciduous Ilex species living on one conti- the latter only few species occur. It is interest- nent are considered to be close to species living to note that deciduous species are only pre- ing on the other continent by Galle (1997). sent in North America and eastern Asia. The This suggests that these species could be sister. 512 BS 55

In the plastid phylogeny, deciduous species are southern hemisphere, whereas it would have limited to Asian/N. American clades I and II decreased it in the northern hemisphere. To (Manen et al. 2002). However, this character correct this artifact due to sampling bias in the does not seem to prevent interspecific southern hemisphere it would be necessary to hybridizations between deciduous and ever- reverse monthly data in the northern hemi- green species. Thus Galle (1997) observed pos- sphere, i.e. to make it coincide between the sible natural hybridisations in seedling beds seasons of both hemispheres. between two North American species: I. decidua Ilex is a genus represented by plants growing Walter (deciduous) and I. opaca Aiton (ever- in relatively wet places and is mostly found in green). forests (Martin 1977). Even if Ilex sometimes grows in drier areas, e.g. on tops of hills in the Potential distribution centre of primary forests, or in secondary forest The current distribution of Ilex seems to be formations, or in savannas, the genus is always directly related to precipitation, minimum near water (forest gallery, swamp). Thus Ilex temperatures and number of frost days. The seems to be able to survive in drier types of veg- projection of the world potential distribution etation if it receives a water complement. As a calculated on the basis of tropical American consequence even if the potential distribution occurrences (Fig. 3) gives a map which largely is more restricted than the extant one, particu- resembles the current distribution of Ilex, if lar local climatic conditions approaching opti- one considers a potential distribution between mal ones would allow the genus to survive in ar- 20% and 100% of habitat suitability. However, eas indicated as unfavourable at the resolution differences are observed in Europe and South of the map. These particular climatic condi- Africa, which have a potential area of distributions cannot appear on a potential map of dis- tion smaller than actual, and along the west tribution using 0.5° square pixel. These micro- coast of Canada, southern coast of Chile, New climatic conditions could explain the occur- Zealand and eastern coast of Australia, where rences of Ilex where the ecological niche proba- the potential distributions are larger than the bility is low. However, it seems that the thresh- extant ones. old of 20% of habitat suitability is the lower lim- Because of the differences in seasonality it in our modelled pattern to find an Ilex plant. between the northern and the southern hemi- Our modelled pattern corresponds rather spheres, only the monthly averages of the well to the reality of the potential distribution month which expresses an annual limit of a of the genus. It requires improvements by given climatic variable were considered. Thus introducing more events, particularly in the for example it is the monthly average of the northern hemisphere and temperate areas, coldest month for each point independently and by adding other climatic variables. How- through all the twelve months which is used to ever, general differences between the model establish the layer of the minimum tempera- and the observed distributions could also be ture. Our model could have been more precise the result of earth’s climatic history which, will considering each month as a variable. But as be discussed further. our presence data cover primarily the tropics of Central and South America, a model using Migration all monthly averages would have increased the Because the fruit of Ilex is adapted to bird dis- area of potential distribution for the habitat persal and the embryo can take between 2 to 8 suitability values between 80% and 100% in the years to germinate (Galle 1997), conditions for BS 55 513 successful long-distance dispersal are met. Tak- firmed, nevertheless, one must wonder how ing into account the study of Myking (2002) on this genus could have been so widespread at the dissemination of Ilex aquifolium L., the that time, since the continents were already genus Ilex would take 100,000 years to migrate quite separate. around the earth under good conditions by A first assumption would be that the genus terrestrial pathways. Bird-effected dispersal Ilex is even older, and that its origin goes back highly accelerates this speed, and moreover to the time when the continents were still makes it possible to place the seeds directly united (early Cretaceous). Willis and McElwain into favourable biotopes, since the birds tend (2002) in particular place the origins of the to seek similar climatological conditions and angiosperms approximately 140 mya. It is biotopes throughout their migration. Crossing strongly unlikely that the genus Ilex, as recog- oceans can be achieved by birds in a few days nised today, was already present at that time. (Schlüssel pers. comm.). Alerstam (1990) indi- APG (1998) places the Aquifoliaceae in a basal cated a 20-hour, non-stop flight to cross the position of the Asterideae, which suggests a Gulf of Mexico (1000 km) for the Turdidea much more recent appearance of this family (typical Ilex fruit dispersors) in their migration within the angiosperm phylogeny. way from United States to Yucatán. In addition, The current distribution pattern of the the probability that a seed carried by the genus Ilex and the available palynologic data marine currents can cross is weak but exists. suggest an Arcto-Tertiary origin of the family. The current distribution of diversity centres Paleogeography would thus be directly related to the evolution Broad distribution of pollen of Ilex at the end of paleo-environmental conditions. The genus of the Cretaceous and throughout the Tertiary would have disappeared from the continents suggests that this genus was widespread at that which would have undergone cooling events time. Projection of pollen fossil occurrences during Miocene until the glaciations of the on paleo-environmental maps drawn from Quaternary. The study of paleo-environmental Beerling and Woodward (2001) (data not models (Beerling & Woodward 2001) lead us shown) showed that in the late Cretaceous, as to believe that no climatic variation, besides in the Eocene, Ilex was found in zones sup- that of the Quaternary between 25,000 and posed to have at least 20° C minimum averages 15,000 years ago could have been the cause of annual temperature and monthly precipitation the extinction of some species of Ilex from cer- average of 100-125 mm. These are coarse data tain areas of the world, e.g. Europe, Africa or and thus represent only tendencies in tempera- New Zealand, where specific diversity is very ture and rainfall variables. But they correspond low for the first two areas and null for the last relatively well to the ecological preferences (no extant Ilex in New Zealand). Paleo-environ- observed for extant species. mental considerations are discussed further, The oldest Ilex pollen, from Australia, was and references are drawn from Adams (1997) dated to be from 90 mya. There are five and Adams and Faure (1997). records of Ilex in the Cretaceous, which are dis- Eurasia – In the Tertiary, pollen of Ilex was tributed in Africa, southeastern Australia, found throughout Asian continent and the northwestern Borneo, California and former genus was widespread from Europe to China. U.S.S.R. These data do not give any indication During the Quaternary, conditions all over on the place of origin of the genus. Although northern Eurasia appear to have been dry and data on Cretaceous Ilex fossils should be con- the continent was treeless. It was dominated by 514 BS 55 polar desert or semi-desert steppe-tundra. very dry, and it is supposed that Ilex was mostly These conditions also extended towards the absent from the continent. Only four species south in Europe, western and central Asia. In are found in Europe (including the Canary both tropical and temperate southern Asia Islands, Madeira and Azores) today, Ilex conditions were much drier and colder than canariensis Poir., Ilex colchica Pojark., Ilex perado now, with diminished areas of forests and Aiton, and Ilex aquifolium L., which are all expanded areas of desert. It thus seems that closely related. A common ancestor could have these climatic conditions made it possible for been the only survivor of the glaciations. It may Ilex to survive in certain areas. The climatic have found refuge in the south of Spain changes did not completely extinguish the and/or in the Canary Islands. If conditions extant species, but instead probably isolated excluded its survival during the Quaternary its them in refugia. This isolation may be the presence could be the result of a recolonisa- cause of the large number of species in south- tion. The four species present in Europe, eastern and eastern Asia today. Isolation could although they are quite isolated from each create conditions for allopatric speciation other are nevertheless relatively close to each events without a complete genetic separation other in their morphology. This could mean of the species, which would maintain the that they result from a unique taxon that was potential of inter-specific hybridisation in the widespread during the Tertiary and which case of Ilex. With later climatic warming the would have survived in disjunct areas during taxa would again come into contact and the Quaternary. hybrids appeared. South-eastern Asia became North America – During the Tertiary, fossil the region which offered the most refugia dur- Ilex pollen was present on the North American ing the successive cooling periods of the Qua- continent. The presence of the Quaternary ice ternary. It has been considered as the center of sheet implies that Ilex was absent from all over origin for Ilex by Hu (1967), but this has not northern North America, nevertheless some been confirmed by the palynological studies species may have existed in refugia in the which show Ilex was already widespread (Mar- forests of the south-east (Webb & Overpeck, tin 1977). http://web.ngdc.noaa.gov/paleo/image/- The high specific diversity in this area could gsafinal.gif). Availability of refuge areas could have two causes which do not exclude each be an important factor in the diversification of other: 1) the rate of extinction in the genus the species and therefore, could explain the was weaker in south-eastern Asia than in other absence of Ilex in western North America areas of the world due to greater availability of despite a potential distribution along the west- refugia during the Quaternary and 2) a greater ern coast of Canada. number of sister species were derived by Central and South America – Central America allopatric speciation during periods of expan- was formed relatively recently (Burnham & sion and regression of refugia and later by Graham 1999) and no Ilex pollen older than 10 hybridization of taxa when they came back into mya has been found. On the other hand, in contact. South America, three Ilex pollen stations of 40 During the Tertiary, Europe was covered by mya in the north and 60 mya in the south have an Arcto-Tertiary flora which included Ilex. A been found. We can conclude that the genus lot of Ilex fossil pollen grains have been found was present in South America before the junc- from this period. During the Quaternary, cli- tion with North America was formed. A signifi- matic conditions were extreme, very cold and cant event on the South American landmass is BS 55 515 the uplift of the Andes. The presence of Ilex for tropical forests, and in drier areas for pollen at the beginning of the uplift of the gallery-forests, existed. In spite of that, the Andes allows us to suppose that the genus genus is represented only by one species, I. shifted up with the uplift of the Andes. Gradu- mitis (L.) Radl. This single species is found in ally climatic conditions changed and some the southern part of the continent south of populations were isolated in valleys by high equator, excluding the desert regions of south- mountains, allowing for allopatric speciation. western Africa. It is also present in Madagascar. It seems that Quaternary cooling did not A first hypothesis could be that Ilex was not have such an important influence on Central widespread in Africa during the Tertiary and and South America as it did on Europe and that it was only present in areas that underwent North America. Central and South America the strongest climatic variations during the probably had refugia in the tropical forests, Quaternary. Another hypothesis would be that such as in Panama and the Amazonian basin. Ilex species were widespread during the Ter- The connection between the Andes and Cen- tiary, but species were not isolated in refugia tral America probably allowed the Andean during the cooling periods and they were species, driven out by unfavourable climatic almost eliminated at that time, and/or later, conditions in the Quaternary, to take refuge in during the cooling periods of the Quaternary. less arid areas of Central America. The Andean The genus would finally almost disappear from uplift and the extensions and regressions of this continent, Ilex mitis being the only species favourable habitats during the Quaternary and to survive. the absence of desertification probably sup- But if all species of Ilex were eliminated from ported specific diversity of Ilex in this region. Africa, its presence could still be the result of a Australia and New Zealand – Ilex pollen from subsequent colonisation. The fact that I. mitis is southern and eastern Australia has been found well established in Madagascar could also sug- from the Tertiary and in New Zealand from the gest a colonization of continental Africa from middle Tertiary. During the Quaternary, this island. The Malagasy flora seems to pass species took refuge in the northern Australia more easily from the island to the African con- (Martin 1977). At that time the centre of Aus- tinent than the opposite. In addition, the ori- tralia became a desert and the south-eastern gin of a great number of Malagasy species forest went extinct. A bridge connected Aus- could be Asia, due to favourable marine cur- tralia to New Guinea. Some rain forests proba- rents which cross the Indian Ocean and which bly survived in New Guinea and in the far would support long distance dispersal (Gautier north of Australia implying potential refugia pers. comm.). This could explain why I. mitis is for Ilex. In New Zealand forests probably disap- close to a certain number of Asian species in peared completely leading to the extinction of the nuclear and plastid clades (Manen et al. the genus in these islands. 2002). Additional studies are needed. Africa – It is surprising to note that very few Ilex pollen grains have been identified from the Tertiary of Africa. Only two occurrences are Conclusions known, dated 85 mya and 5 mya, respectively. The genus Ilex was probably widespread from Nothing indicates that the climate during the the beginning of the Tertiary, and perhaps Tertiary or Quaternary in Africa could have led before. The first Ilex pollen fossils are dated to the extinction of the genus. Indeed, during older than 85 mya, and were found in Africa, the coldest period of the Quaternary refugia Australia and Asia. Consequently it is impossi- 516 BS 55 ble to have an idea of the origin of the genus records in our database (more than 3500 indi- on the basis of palynological studies. In addi- viduals from Central and South America) tion, a calculation based on the molecular made it possible to propose a potential distrib- clock for the extant Ilex species gives an origin ution of the genus Ilex. The potential distribu- of approximately 50 mya. This is interpreted as tion corresponds, in the broad outline, to the being the age of the oldest ancestor of the cur- extant distribution except for some areas in rent species. Any trace of even older ancestors western North America and in Africa. The is lost with the extinction of the branches assumption made is that the genus was wide- which corresponded to them. spread at the end of the Tertiary and that it has The study of the extant species shows a great been restricted to the current distribution due homogeneity of floral morphology within the to colder and drier climatic conditions during genus in spite of, according to the molecular the Quaternary. The very high number of clock, approximately 50 million years of evolu- species in south-eastern Asia and South Amer- tion. This fact suggests a “morphological sta- ica could be directly related to these two areas sis,” that is to say a great stability of characters acting as refugia during the Quaternary, and to over a very long time period. In addition, it the slow uplift of the Andes during the Tertiary seems that different species hybridize leading in South America. up to morphological and molecular homo- The situation seen in Africa is not clear. geneisation. This apparent great stability of Either Ilex was never widespread on this conti- characters and the possibility of hybridisation nent during the Tertiary, or it was widespread makes the elucidation of relations between but climatic conditions of the Quaternary were species even more difficult. more drastic than currently knowledge sup- Attempts to reconstruct phylogenies of poses implying widespread extinction in Africa extant species based on sequencing of plastid (Raven & Axelrod 1974). and nuclear DNA, confirm the potential to To improve our knowledge of Ilex distribu- hybridize. A phylogeny based on morphologi- tion patterns, it will be necessary to work on cal characters was not resolved and brought no several levels: new information to the problem. Comparison Complete the pollen data, particularly for between morphological characters and plastid South America and Africa. In addition, it phylogeny, which seemed to give the most sig- would be interesting to have information nificant results compared to the nuclear one, about the presence of Ilex in the Antarctic dur- showed that there was no correlation between ing the Tertiary (Partridge pers. comm.). them. All the above mentioned factors high- Improve our knowledge of the pattern of light the enormous difficulty encountered potential distribution by integrating data from when attempting to connect the evolutionary temperate zones of the northern hemisphere, history of the genus with the extant distribu- and increasing the number of climatic vari- tion, and in determining centres of origin for ables. This will better define the ecological the genus. Information currently available can- niche and allow us to compare it to paleoenvi- not give any indication of the number of ronmental patterns. species present in the past, and it gives only a Improve our knowledge of species variability. few indications of possible migratory pathways This will allow us to have a better understand- of these species according to extant distribu- ing of the different taxa and clarify better their tions. current distribution patterns. A statistical analysis based on specimen BS 55 517

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Appendix 1: The 47 Ilex species selected for the morphological phylogeny. Descriptions were based on cited literature and speciminens seen which are indicated with the collector, the collector’s number, the acronym of the herbarium and their sexual status (f: female, m: male, st: sterile).

Taxa Literature and Specimens seen I. amelanchier M. A. Curtis Loesener 1901, Galle 1997, Drummond s.n. 1832 BM f, Leonard 1728 BM f, Curtis s.n. 1852 G f I. anomala Hook. & Arn. Loesener 1901, Galle 1997, Faurie 282 G mf, Degener 20061 G f, Degener 3316 G m, Degener 3324 G m, I. argentina Lillo Galle 1997, Beck 9660 G m, Venturi 4623 S m, Giberti 508 G f, Venturi 9990 S f I. brasiliensis (Sprengel) Loes. Giberti 1994, Regnell II 56 S m, Mosén 4243 S f, Mosén 1822 S f I. brevicuspis Reissek Giberti 1994, Balansa 1793 S f, Hatschbach 30782 MU m I. canariensis Poir. Loesener 1901, Galle 1997, Mandon s.n. 1865-1866 G m, Mason 349 G f, Asplund 1112 G m I. cassine L. Galle 1997, Standley 190 BM f, Tracy 6829 BM mf, Drummond s.n. BM st, Curtiss 1747 BM m I. crenata Thunb. Loesener 1901, Galle 1997, Anonymous 155 G m, Kasapligil 3660 G f, Faurie 3114 G m I. decidua Walter Loesener 1901, Galle 1997, Ventenat s.n. G m, Godfrey 54470a G m, Eisenbeiss & Dudley s.n. 1979 G f, Bosc s.n. G f, Godfrey 54549 G f I. dumosa Reissek Giberti 1994, Schinini 31582 G m, Hatschbach 22945 S m, Schinini 31420 G f I. glabra (L.) A. Gray Galle 1997, Bartram 445 BM mf, Bray 8423 BM m, Rugel 3365 BM f I. goshiensis Hayata Galle 1997, Furuse 2498 G m, 2768 K f, 2959 K m, 3550 K f I. guianensis (Aubl.) Kuntze D’Arcy & al. 15500 G m, 15506 G m, Churchill 4284 G m, Gentry & al. 47534 COL m, 47555 COL f, Werff, van der & al. 6153 G f, 6916 G m I. hippocrateoides Kunth Humboldt & Bonpland s.n. P m, Núñez & al. 9034 G st, 9904 G f, Hamilton & Holligan 661 K st, Smith 2723 G f I. integerrima (Vell.) Reissek Loesener 1901, Freyreis s.n. S f I. latifolia Thunb. Loesener 1901, Galle 1997, Maximowicz s.n. G mf I. laurina Kunth Bernardi 1045 NY m, Sneidern 4356 F m, Lehmann B.T.668 G, NY m, B.T.959 NY f, Williams 6995 COL, F f, Cuatrecasas 18281 F, G, US f, Humboldt & Bonpland s.n. W m I. leucoclada (Maxim.) Galle 1997, Wilson 7099 BM mf, Wilson 7630 BM f Makino I. liebmannii Standl. Standley 1931, Galle 1997, Liebmann 14927 G f (fragments). I. longipes Trel. (= I. collina) Loesener 1901, Galle 1997, Nogle s.n. 1956 BM f, Dudley s.n. 1976 BM m, K f I. macrocarpa Oliver Loesener 1901, Galle 1997, Linsley Gressitt 1736 G f I. macropoda Miq. Loesener 1901, Galle 1997, Tschonoski s.n. 1864 G mf, Watari s.n. 1951 G f, Anonymous s.n. 1888 G m, Nagamasu 5457 G m I. maximowicziana Loes. Loesener 1901, Galle 1997, Furuse 3400 G m, Furuse 1060 K f, Furuse 2883 K f,. I. micrococca f. pilosa S. Y. Hu Galle 1997 (I. micrococca), Henry 11974a K f, Fang 5656 K f, Forrest 8651 K f, 15749 K m I. microdonta Reissek Hatschbach 24206 S f, 22837 COL m I. mitis (L.) Radlk. Galle 1997, Pegler 1366 BM m, Zeyher 1129 BM m, Bolus 5202 BM mf, Drege s.n. BM mf I. mucronata (L.) M. Powell, Loesener 1901, Chapman s.n. 1844 G m, Pringle s.n. 1877 G f, Cinq-Mars 67-54 G m V. Savolainen & S. Andrews 520 BS 55

Taxa Literature and Specimens seen I. mutchagara Makino Furuse 4812 K m I. oppositifolia Merrill Galle 1997, Clemens 31108 BM f, 31375 BM st, 32249 BM f, 40539 BM m I. pedunculosa Miq. Galle 1997, To Hara 2441 K m, Maximowicz s.n. K mf, Murata 15833 K f I. perado Aiton Galle 1997, Loesener 1901, Reverchon 76 G f, Mason 343 G st I. pseudobuxus Reissek Loesener 1901, Mosén 2898 S m, 3651 S f, Britez 1397 MBM f, Tessmann s.n. 1953 MBM m I. pubescens Hook. & Arn. Loesener 1901, Galle 1997, Shiu Ying Hu 8996 G f, Tso 20100 G m, Lingnan 12016 G f I. purpurea Hassk. Loesener 1901, Galle 1997, Chiao 1683 G f, Nagamasu 5516 G f, 5524 G m I. repanda Griseb. Wright 1142 BR f, Wright 1142 K mf, Curtiss 88 K mf, Jack 4835 K f I. revoluta Stapf Galle 1997, Clemens s.n. BM m, 32315 BM m, Gibbs 9127 BM f I. rotunda Thunb. Galle 1997, Wright 184 K mf, Furuse 2695 K m, Furuse 2939 K f I. rugosa F. Schmidt Loesener 1901, Galle 1997, Yatabe s.n. 1882 G f, Tschonoski s.n. 1864 G mf I. serrata Thunb. Loesener 1901, Galle 1997, Maximowicz s.n. 1862 [Fudziyama] G f, Maximowicz s.n. 1862 [Yokohama] G f, Franchet 178 G m I. shennongjiaensis Dudley & Sun 1983, Galle 1997 T.R.Dudley & S.C. Sun I. sugerokii Maxim. Galle 1997, Wilson 7100 K mf, Fukuoka 1052 K m, Maximowicz s.n. K f, Wilson 7183 K f I. teratopis Loes. Pearce s.n. K mf I. theezans Reissek Giberti 1994, Dusén 15543 S m, Oliveira 682 MU f, Dusén s.n.1909 S f, Francisco & al. s.n.1999 G m I. triflora Blume Galle 1997, Kerr 13277 BM f, 15523 BM f, 17771 BM m, Tsang 29153 G m, Taam 675 G m I. verticillata (L.) A. Gray Galle 1997, Anonyme s.n. [1824] BM m, Euphrosin s.n. [1926] BM m, Judd s.n. [1932] BM f, Bauers 4 K f I. vomitoria Aiton Galle 1997, Rugel 92 BM mf, Hood 1996 BM f, Miller 9460 BM f I. yunnanensis Franch. Hu 1949, Galle 1997, Delavay 2673 K m, Forrest 10247 K m, Fliegner 1193 K f