Distribution and variation of sexual and agamospermous populations of Stevia (Asteraceae: Eupatorieae) in the lower latitudes, Mexico

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1 Plant Species Biology (2001) 16, Distribution and variation of sexual and agamospermous populations of Stevia (Asteraceae: Eupatorieae) in the lower latitudes, Mexico AKIKO SOEJIMA,* TETSUKAZU YAHARA and KUNIAKI WATANABE *College of Integrated Arts and Sciences, Osaka Prefecture University, Sakai , Department of Biology, Faculty of Science, Kyushu University, Fukuoka and Department of Biology, Faculty of Science, Kobe University, Kobe, Japan Abstract Cytogeographical studies have revealed the distribution of sexual diploids and agamospermous polyploids of Stevia ovata and four related species: Stevia nepetifolia, Stevia oligophylla, Stevia origanoides and Stevia triflora in Mexico. These species are distributed mainly in the temperate and subhumid montane region. Agamospermous triploids are the forms most abundantly and widely distributed geographically in each of the five species. Diploids occur in low frequencies except in S. origanoides. For the five species, diploids are restricted mainly to the temperate montane ranges of the south-western Pacific side of Mexico, but they also occur sporadically in the relatively isolated areas from Oaxaca to Durango, Mexico D. F. and Nuevo Leon. Although the distribution of the temperate forests must have moved southward and northward following climatic fluctuations, even in the lower latitudes of Mexico, the largely stable temperate vegetation could have been maintained along the Y-shaped montane ranges (Sierra Madre del Oriental, Sierra Madre del Occidental, Sierra Madre del Sur and the Trans-Mexico Volcanic Belt) and have provided some refuge areas. The present distribution ranges of sexual diploids of some species of Stevia in Mexico indicate that diploids were not restricted to a single refuge during the last ice age. Keywords: agamospermy, Asteraceae, cytogeography, polyploidy, Stevia. Received 13 October 1999; revision received 12 October 2000; accepted 1 November 2000 Introduction Within the large genus Stevia, many species are known to have both sexual and agamospermous forms. In Mexico, agamospermy accompanied by polyploidy seems to be a fairly common mode of reproduction among the perennial herbaceous species of Stevia (Grashoff 1972, 1974; Turner 1997). Stevia ovata Willd. is one of the most widely distributed species of this genus in North and Central America, ranging from Texas southward to Ecuador. According to Grashoff (1972), sexual forms of S. ovata are isolated and scattered throughout Mexico; therefore, it is suitable material to examine the distribution of a sexualagamospermous complex in relation to sexuality and historical changes in a distributional pattern. This species Correspondence: Akiko Soejima soejima@el.cias.osafu-u.ac.jp shows wide morphological variation, and intermediate forms with other species are sometimes found. Among such morphologically related species, Stevia nepetifolia H.B.K., Stevia oligophylla Soejima & Yahara, Stevia origanoides H.B.K., and Stevia triflora DC. all have relatively large distribution areas that overlap that of S. ovata. Stevia origanoides and S. triflora are also known to be sexual-agamospermous species (Grashoff 1972). There have been many studies on the distributional pattern of sexual-agamospermous complexes: Antennaria (Bayer & Stebbins 1983), Boehmeria (Yahara 1986), Crepis (Babcock & Stebbins 1938), Eupatorium (Watanabe 1986; Kawahara et al. 1989), European blackberries (Gustafsson 1943), and Taraxacum (den Nijs & Menken 1996). Most of these studies were carried out on plants in North America and Europe, where the vegetation of the present temperate regions has been largely influenced by climatic cooling and/or glaciation during the last glacial period. The dis The Society for the Study of Species Biology

2 92 A. SOEJIMA ET AL. tributional patterns of sexual-agamospermous complexes have been interpreted based on past distributions of glaciated unglaciated areas and the north to south migrations of flora during the glacial and interglacial periods. Agamospermous forms tend to occur in glaciated areas, and sexual forms are commonly restricted to a few small areas around unglaciated refuges at higher latitudes (Bierzychudek 1985, 1987; den Nijs & Menken 1996). However, there are few studies on sexual and agamospermous conspecific populations in lower latitudes. Mexico, located between the latitudes N and N, served as one of the major refuges for flora in North America during the last glacial period (Martin & Harrell 1957). Although little is known about the flora of Mexico during the last ice age (Rzedowski 1981), the influence of glaciers is considered to have been significant only above an altitude of 3300 m (Tamayo 1995). Stevia in Mexico currently occur from altitudes approximately 1000 to 3000 m; thus, the distributional patterns of sexualagamospermous complexes in this genus could differ from complexes in the higher latitudes. To elucidate the distributional pattern of this sexualagamospermous complex in the lower latitudes of Mexico, we examined the chromosome number and the morphological variation of leaves of five species in Mexico: S. nepetifolia, S. oligophylla, S. origanoides, S. ovata and S. triflora. Materials and methods Plants collected in Mexico and the USA (in Texas) were cultivated in Osaka, Japan. Cytological observations were made on mitotic root tip cells. Root tips were collected and kept in ice water for 24 h, and fixed in ethanol : acetic acid (3 : 1) for at least 15 min. Maceration and staining were carried out simultaneously in 1% aceto-orcein : 1N HCl (9 : 1) at room temperature for more than 12 h before squashing. Voucher specimens are deposited in MEXU and KYO. Results Chromosome number and distribution The chromosome numbers of 198 accessions were determined (see Appendix I). The observed chromosome numbers were 2n = 22, 23, 24, 25, 26, 33, 34, 35, 36, 44, 45, 46, 47, 48, 55, 60 and 66. Watanabe et al. (2001) reported the frequent occurrences of B chromosomes in Stevia, and found a maximum of eight B chromosomes in one cell. The B chromosomes of Stevia are usually smaller and show more dense condensation than autosomes at prometaphase. However, it is often difficult to distinguish B chromosomes from autosomes, and a chromosome is referred to as B in this paper only when the two classes were distinctly recognized. According to Watanabe et al. (2001), the basic chromosome number is x = 11 and 12 in perennial Stevia. Thus, plants with 2n = 22, 23, 24, 25 or 26 may be regarded as diploids; plants with 2n = 33, 34, 35 or 36 as triploids; plants with 2n = 44, 45, 46, 47 or 48 as tetraploids; plants with 2n = 55 and 60 as pentaploids; and plants with 2n = 66 as hexaploids. We found polyploid series in each of the five species studied. Grashoff (1972) reported no sexual plants in S. nepetifolia based on pollen observation. However, five diploids were found during the present study in the states of Guerrero and Oaxaca. The distribution of diploids and polyploids for each species are shown in Fig. 1. Triploids were the predominant forms and were abundant in each of the five species studied. In S. nepetifolia, S. oligophylla, S. ovata and S. triflora, diploids occurred in various restricted areas mainly in the mountain regions on the south-western Pacific side of Mexico. Some diploid S. ovata were also found in the central mountains (Mexico D. F.) and in Sierra Madre del Oriental in Nuevo Leon, the north-eastern part of Mexico. Diploids of S. origanoides occurred more frequently throughout a relatively wide range in the mountains along the Pacific coast. The distributional ranges of the five species overlap considerably with each other, and some of the species were sympatric. Moreover, conspecific plants of different ploidy levels sometimes occurred within a given population (see Appendix I). Morphological variation Each of the species studied showed a range of variation in morphology and plant size. Plant size did not seem to be correlated with ploidy level; diploids were not always smaller than their polyploid relatives. The range of morphological variation differed from species to species. Most plants could be identified as one of the five species. However, occasional plants showed morphologically interspecific or extraordinary forms, and were omitted from this study. Stevia nepetifolia The five diploids of S. nepetifolia had relatively small, elliptic to rhombic and densely white-haired leaves (Fig. 2). However, hair density and leaf size varied continuously among polyploid individuals (Fig. 2). Some plants of this species, with lower hair density and larger leaves, appear to be similar to S. ovata, although we could distinguish between the two species in most cases. Stevia oligophylla This is a newly recognized species that is easily distinguishable as it has fewer and thicker leaves than other

3 DISTRIBUTION AND VARIATION OF STEVIA 93 (a) (d) (b) (e) (c) Fig. 1 Distribution of diploids and polyploids of (a) Stevia nepetifolia; (b) Stevia oligophylla; (c) Stevia origanoides; (d) Stevia ovata; 1. var. expansa, 2. var. reglesens, 3. var. texana; (e) Stevia triflora. ( ) diploid; ( ) triploid; ( ) tetraploid; ( ) pentaploid; (*) hexaploid.

4 94 A. SOEJIMA ET AL. (a) (b) (c) (a) (b) Fig. 2 Leaf shape variations in Stevia nepetifolia. (a) Yahara & Soejima 268 (2 ); (b) Yahara & Soejima 338 (3 ); (c) Yahara & Soejima 837 (3 ). Bar, 1 cm. Fig. 3 Leaf shape variations in Stevia oligophylla. (a) Yahara et al (2 ); (b) Yahara et al (4 ). Bar, 1 cm. species of Stevia (Soejima et al. 2001). We found only one diploid that was morphologically indistinguishable from the polyploids (Fig. 3). Stevia origanoides. Sixteen diploids out of 58 S. origanoides plants examined occurred widely from Chihuahua to Oaxaca (Fig. 1c). The plants of this species are profusely branched and have rather small clusters of heads, but the size and the shape of the leaves are variable. Based on the leaf characters, we could operationally recognize four morphological groups among the diploids, although the morphological variations between them were continuous. These variations were designated as origa-1 to origa-4 (Figs 4 & 5); origa-1 had elliptic leaves with cuneate bases, origa-2 had linear lanceolate leaves, origa-3 had ovate to elliptic leaves with somewhat truncate bases, and origa-4 had larger linear lanceolate leaves and few branches. Most of the triploids and tetraploids were categorized as belonging to origa-1 or origa-2, respectively. Stevia ovata. This species was characterized by relatively large, elliptic to rhombic and nearly glabrous leaves. Six out of the 79 S. ovata plants examined were diploids and were collected from three localities (Fig. 1d). The diploids were rather uniform in leaf shape and branching pattern. One of the diploid populations was identified to be Stevia ovata var. expansa because it had lax inflorescences, but the population was adjacent to a diploid population of the typical variety; var. expansa was found at 1480 m, while var. ovata was found at 1780 m within the same locality (Appendix I). Triploids were the most abundant forms and had the widest geographic distributional ranges. The leaf shape and size of triploids varied continuously, and it was impossible to separate them into morphological groups (Fig. 6). Tetraploids, pentaploids, and hexaploids were less frequent, and their morphological variation ranges seemed to be narrower than that of the triploids. Polyploids of S. ovata tended to be larger in terms of plant size, but the morphological features, such as leaf shape and hair density, were not very different from those of the diploids. Stevia triflora. This species resembled S. ovata, but differed in terms of having obviously yellowish hair on the stems and leaves. Three diploids, eight triploids and five tetraploids were found in the present study. Three diploids were collected from a single locality (Fig. 1e). Diploids and polyploids were morphologically indistinguishable (Fig. 7). Figure 8 shows the relationship between the altitude and latitude of collection localities and the ploidy levels of plants for all species examined. No particular relationships between these attributes could be found. Discussion Both diploids and polyploids were found in each of the five species studied. Sexual diploids of S. ovata and related species mainly occur along the mountain ranges of the south-western Pacific side of Mexico, in the localities such as Sierra Madre del Sur and Sierra Madre del Occidental (Fig. 1). However, S. ovata and related species

5 DISTRIBUTION AND VARIATION OF STEVIA 95 (a) (b) (c) (d) (e) (f) Fig. 4 Leaf shape variations in Stevia origanoides. (a) Yahara et al. 814 (2 ; origa-1); (b) Yahara et al 785 (2 ; origa-2); (c) Yahara & Soejima 297 (2 ; origa-3); (d) Yahara & Soejima 460 (2 ; origo-4); (e) Yahara et al (2 ; origa-3); (f) Yahara et al. 961 (3 ; lanceolate-form). Bar, 1 cm. Table 1 Frequencies of diploids and polyploids; numbers of plants observed and percentages in parentheses Diploid (2 ) Triploid (3 ) Tetraploid (4 ) Pentaploid (5 ) Hexaploid (6 ) Total Stevia nepetifolia 5 (15.2) 19 (57.6) 8 (24.2) 1 (3.0) 0 (0.0) 33 Stevia oligophylla 1 (9.1) 7 (63.6) 2 (18.2) 1 (9.1) 0 (0.0) 11 Stevia origanoides 16 (27.6) 33 (56.9) 9 (15.5) 0 (0.0) 0 (0.0) 58 Stevia ovata 6 (7.6) 55 (69.6) 11 (13.9) 5 (6.3) 2 (2.5) 79 Stevia triflora 4 (23.5) 9 (52.9) 4 (23.5) 0 (0.0) 0 (0.0) 17 are not restricted to a single area, but also occur in the central high mountain region (Trans-Mexican Volcanic Belt) and north-eastern mountains (Sierra Madre del Oriental). The diploid populations are scattered, and there are no particular relationships between ploidy level and altitude (Fig. 8). The whole distributional area of these species roughly corresponds to the region of Central American Montane Flora, which is under a temperate and subhumid climate (Rzedowski 1981) (Fig. 9). This floristic region consists mainly of pine oak forest, and is one of the richest regions for biological diversity and endemism in Mexico (Graham 1993; Rzedowski 1993). The distributional range of agamospermous polyploids is generally larger than that of ancestral diploids in the higher latitudes of Northern Hemisphere. This is thought to be due to the ability of agamospermous polyploids to migrate rapidly into glaciated areas where temperate vegetation was cleared off or replaced with boreal vegetation in the last glacial period. Based on the fossil records, Soreng & Devender (1989) presented good evidence that the agamospermous races of Poa fendleriana migrated northward during the latest Wisconsin Glacial Age, or the early Holocene, and were followed years later by the sexual races. In the lower latitudes, such

6 96 A. SOEJIMA ET AL. (a) (b) (c) (d)

7 DISTRIBUTION AND VARIATION OF STEVIA 97 (a) (b) (c) (d) (e) (f) (g) (h) (i) Fig. 6 Leaf shape variations in Stevia ovata. (a) Yahara et al. 718 (2 ); (b) Yahara et al. 819 (2 ); (c) Yahara & Soejima 335 (3 ); (d) Yahara et al. 949 (3 ); (e) Yahara et al. 848 (3 ); (f) Yahara et al. 977 (3 ); (g) Yahara et al. 621 (3 ); (h) Yahara et al. 729 (4 ); (i) Yahara & Soejima 281 (5 ). Bar, 1 cm. Fig. 5 Morphological variations in Stevia origanoides. (a) Yahara et al. 472 (2 ; origa-1) Stevia origanoides H.B.K., flowers white; location Jalisco, 12.5 km south of Autlan on Route 80, 0.5 km south of Puerto del Mazos; in edge of oak forest, on cliff, 1400 m; 1 December 1995; collection T. Yahara & A. Soejima, no. 472 (2n = 22). (b) Yahara et al 785 (2 ; origa-2) Stevia origanoides H.B.K.; location Durango, km north-east of Vila Union to Durango on Mex 40, on east-facing cliff, 1960 m, 31 October 1996; collection T. Yahara, A. Soejima, T. Kajita & T. Miyake, no. 785 (2n = ~4B). (c) Yahara & Soejima 297 (2 ; origa-3) Stevia origanoides H.B.K.; location Guerrero, 23.7 km south of Petaquillas on Highway 95 Libre, on the way from Chilpancingo to Acapulco, on half-shaded slope along roadside, 1100 m; 13 November 1995, collection T. Yahara & A. Soejima, no. 297 (2n = ~3B). (d) Yahara & Soejima 460 (2 ; origa-4) Stevia origanoides H.B.K., achenes exaristate; location Jalisco, 11 km east from Junction of Talpa de Allende-La Cuesta route, on the way to El Chilacayote, in an open place on a rock plate around a rocky hilltop, 2220 m; 29 November 1995; collection T. Yahara & A. Soejima, no. 460 (2n = 22). Bars, 5 cm. as in Mexico, the distributional patterns of sexual diploids and agamospermous polyploids are basically the same as those in the higher latitudes. The distribution range of diploids in some species is smaller than that of polyploids; however, diploids sometimes have disjunctively scattered and wide distributions in Mexico. The microfossil record has revealed the existence of cool temperate flora in the southern part of Veracruz in Mexico during the middle Pliocene before the ice age (Graham 1976, 1989, 1993, 1999). This indicates that the mountain regions of Mexico could provide a large refuge and an effective corridor for humid subhumid temperate flora of North America throughout the glacial and interglacial ages (Martin & Harrell 1957; Graham 1993, 1999; Rzedowski 1993). In central Mexico, an orogenic movement uplifted the Trans-Mexican Volcanic Belt through the late Miocene to the Pleistocene. Such physiographical change and climatic undulation during the ice age is supposed to have affected the diversification of Mexican flora (Graham 1993). The temperate forest must have moved

8 98 A. SOEJIMA ET AL. (a) Fig. 7 Leaf shape variations in Stevia triflora. (a) Yahara et al. 943 (2 ); (b) Yahara et al. 757 (3 ). Bar, 1 cm. (b) south and northwards along these mountains following climatic fluctuations, even in Mexico. However, a stable and considerably large core area of temperate forest was probably maintained. The timberline was lowered by about 1000 m during the Wisconsin Glacial Age in southern and northern regions of Mexico (McDonald 1993). In the north-eastern part of Mexico, the distributional limits of the timberline are thought to be approximately 2500 m for glacial periods and m for interglacial periods (McDonald 1993). Thus, the vegetation in most of the current Stevia zone (between 1000 and 3000 m) probably existed through the last glacial period. Moreover, based on pollen analysis of sediment cores, Watts & Bradbury (1982) demonstrated that the vegetation around Lake Patzcuaro (on the western-central Mexican Plateau at 2044 m in the state of Michoacán) has not changed drastically during the last years. Pine oak forests containing abundant fir have existed there continuously until the present. This implies that a more widely distributed (a) (d) Latitude ( ) Latitude ( ) Altitude (m) Altitude (m) 30 (b) 30 (e) Latitude ( ) Latitude ( ) Altitude (m) 30 (c) Altitude (m) Latitude ( ) Altitude (m) Fig. 8 The relationship of ploidy levels and altitudelatitude of collection localities. (a) Stevia nepetifolia; (b) Stevia oligophylla; (c) Stevia origanoides; (d) Stevia ovata; (e) Stevia triflora; ( ) 2 ; ( ) 3 ; ( ) 4 ; (*) 5.

9 DISTRIBUTION AND VARIATION OF STEVIA 99 Fig. 9 Distribution of Central American Montane Flora (from Rzedowski 1981). pine oak forest, which is the main habitat of Stevia, could have existed in a stable condition throughout the last glacial period and the Holocene. The scattered and wide geographic distribution of diploids in species such as S. origanoides may be attributed to the geographically widespread and stable temperate flora that existed during the glacial period. Moreover, the present morphological variation in the ploidy levels of S. ovata and S. origanoides seems to be due to multiple polyploidization events. This suggests that a large geographic distribution of diploids has been maintained in the past. Even though the present distribution areas of diploids are not restricted to a single region, the distribution areas of polyploids are still larger in each of the Stevia species examined in the present study. It is known that some agamospermous races of Stevia of North and Central America, such as S. ovata and S. serrata, extend to the northern parts of South America (Grashoff 1972), which may be a secondary range extension following human activity. The larger distributional range of polyploids, sometimes extending to the subtropical zone, may be due to their rapid colonizing ability. In addition, temperate forests in the past have been destroyed by increasing of human disturbance (e.g. following the Holocene). At Lake Patzcuaro, which is located within a region of temperate vegetation, pollen grains of Zea have been found dating from 3500 years ago to the present. This means that human activity had enough impact to modify the natural environment (Watts & Bradbury 1982) during this time period. In spite of the well-preserved temperate forests during the last glacial period, 30% of the total area of the humid temperate zone and 26% of the subhumid temperate zone are being used for agriculture and livestock at present (Toledo & Ordóñez 1993). This artificial usage may be one of the reasons for the present disjunctive distribution of diploids and larger distribution of polyploids. Because the temperate vegetation was maintained during the ice age in the lower latitudes, diploids of Stevia have also been maintained to a certain scale. This is why some diploid species still have wider distribution ranges within relatively isolated areas and wider variation in morphologies in Mexico than would be expected at the higher latitudes. Acknowledgements We are grateful to Drs Jose Luis Villaseñor, Ken Oyama, Jose Panero, and B.L. Turner for providing us with useful information and support during our field trips and herbarium works in MEXU and TEX. Thanks a lot to Drs T. Kajita, K. Ooi, M. Mishima, and T. Miyake for helping and encouraging us in the field trips. A Grant in Aid for International Scientific Research (Field Research) no to Tetsukazu Yahara and a Grant in Aid for

10 100 A. SOEJIMA ET AL. Scientific Research no to Tetsukazu Yahara from the Ministry of Education, Science and Culture, Japan, supported this study. References Babcock E. B. & Stebbins G. L. (1938) The American species of Crepis. Their interrelationships and distribution as affected by polyploidy and apomixis. Publisher of Carnegie Institution, 504: Bayer R. J. & Stebbins G. L. (1983) Distribution of sexual and apomictic populations of Antennaria parlinii. Evolution 37: Bierzychudek P. (1985) Patterns in plant parthenogenesis. Experientia 41: Bierzychudek P. (1987) Patterns in plant parthenogenesis. In: The Evolution of Sex and its Consequences (ed S. C. Stearns) pp Birkhauser-Verlag, Basel. den Nijs H. C. M. & Menken S. B. J. (1996) Relations between breeding system, ploidy level, and taxonomy in some advanced sections of Taraxacum. In: Compositae: Systematics. Proceedings of the International Compositae Conference, Kew, 1994 Vol.1 (eds D. J. N. Hind & H. J. Beentje) pp Royal Botanic Gardens, Kew. Graham A. (1976) Studies in Neotropical paleobotany. II. The Miocene communities of Veracruz, Mexico. Annals of the Missouri Botanical Gardens 63: Graham A. (1989) Paleofloristic and paleoclimatic changes in the Tertiary of northern Latin America. Review of Paleobotany and Palynology 60: Graham A. (1993) Historical factors and biological diversity in Mexico. In: Biological Diversity of Mexico. Origins and Distribution (eds T. P. Ramamoothy, R. Bye, A. Lot & J. Fa) pp Oxford University Press, New York. Graham A. (1999) The Tertiary history of the northern temperate element in the northern Latin American biota. American Journal of Botany 86: Grashoff J. L. (1972) A systematic study of the North and Central American species of Stevia. PhD Dissertation, University of Texas (unpubl.) Austin. Grashoff J. L. (1974) Novelties in Stevia (Compositae: Eupatorieae). Brittonia 26: Gustafsson A. (1943) The genesis of the European blackberry flora. Lunds Universitets Arsskrift N. F. Avd. 239: Kawahara T., Yahara T. & Watanabe K. (1989) Distribution of sexual and agamospermous populations of Eupatorium (Compositae) in Asia. Plant Species Biology 4: Martin P. S. & Harrell B. E. (1957) The Pleistocene history of temperate biotas in Mexico and eastern United States. Ecology 38: McDonald J. A. (1993) Phytogeography and history of the alpinesubalpine flora of northeastern Mexico. In: Biological Diversity of Mexico Origins and Distribution (eds T. P. Ramamoothy, R. Bye, A. Lot & J. Fa) pp Oxford University Press, New York. Rzedowski J. (1981) Vegetacion de Mexico. Limusa Noriega Editores, Mexico D. F. (In Spanish.) Rzedowski J. (1993) Diversity and origins of the phanerogamic flora of Mexico. In: Biological Diversity of Mexico. Origins and Distribution (eds T. P. Ramamoothy, R. Bye, A. Lot & J. Fa) pp Oxford University Press, New York. Soejima A., Yahara T. & Watanabe K. (2001) Thirteen new species and two new combinations of Stevia (Asteraceae: Eupatorieae) from Mexico. Brittonia 53 (in press). Soreng R. J. & Van Devender T. R. (1989) Late Quaternary fossils of Poa fendleriana (Muttongrass): Holocene expansions of apomicts. Southwestern Naturalist 34: Tamayo J. L. (1995) Geografia moderna de Mexico. Trillas, Mexico D. F. Toledo V. M. & Ordóñez M. J. (1993) The biodiversity scenario of Mexico: a review of terrestrial habitats. In: Biological Diversity of Mexico. Origins and Distribution (eds T. P. Ramamoothy, R. Bye, A. Lot & J. Fa) pp Oxford University Press, New York. Turner B. L. (1997) The Comps of Mexico. A systematic account of the family Asteraceae Vol. 1 Eupatorieae. Phytologia Memoirs 11: Watanabe K. (1986) The cytogeography of the genus Eupatorium (Compositae): A review. Plant Species Biology 1: Watanabe K., Yahara T., Soejima A. & Ito M. (2001) Mexican species of the genus Stevia (Eupatorieae, Asteraceae): Chromosome numbers and geographical distribution. Plant Species Biology 16: Watts W. A. & Bradbury J. P. (1982) Paleoecological studies at Lake Paztcuaro on the west-central Mexican Plateau and at Chalco in the basin of Mexico. Quaternary Research 17: Yahara T. (1986) Distribution of sexual and agamospermous populations of Boehmeria sylvestrii and its three relatives (Urticaceae). Memoirs of the National Science Museum 19:

11 Appendix I. Chromosome number determinations in Mexican Stevia DISTRIBUTION AND VARIATION OF STEVIA 101 Species and locality 2n Ploidy level Stevia nepetifolia H.B.K. Chiapas, 66 km NE of San Cristobal de Las Casas, on Hwy 186, 1260m. Yahara et al Chiapas, 4.8 km NW of Motozintla de Mendoza, on the way to Siltepec, 1610 m. Yahara et al Durango, 14.7 km W of Suchil, on the way to San Miguel de la Michilia, 2070m. Yahara et al Durango, 42.1 km SW of San Juan de Michis, 4.3 km W from JCT to Las Margaritas, 2505 m. Yahara et al Durango, 12 km S of Mezquital, on the way from San Juan de Michis to Mezquital, 2120 m. Yahara et al Guerrero, 42 km SW from JCT of Hwy 95 Libre to Filo de Caballo, 2220 m. Yahara & Soejima Guerrero, 49 km SW from JCT of Hwy 95 Libre to Filo de Caballo, 2200 m. Yahara & Soejima Guerrero, 8.5 km SW from Filo de Caballo, 63.5 km SW from JCT of Hwy 95 Libre, 2540 m. Yahara & 36 3 Soejima 283 Guerrero, 8.5 km SW from Filo de Caballo, 63.5 km SW from JCT of Hwy 95 Libre, 2540 m. Yahara & 36 3 Soejima 284 Guerrero, Cerro Alquitran, on the way from Mazatlan to Microwave station, 2180m. Yahara et al Guerrero, 42km SW from Hwy 95, on the way from Chilpancingo to Filo de Caballo, 2080m. Yahara 36 3 et al. 857 Guerrero, 48.5km SW from Hwy 95, on the way from Chilpancingo to Filo de Caballo, 2050m. Yahara 25 2 et al. 863 Guerrero, 35.8km SW from Hwy 95, on the way from Chilpancingo to Filo de Caballo, 1160m. Yahara 36 3 et al. 872 Jalisco, 1.6km W of El Pueblito, along Mex 15. Yahara & Soejima ~3B 3 Mexico, 8.8km NE of Mex 134 from San Francisco de los Ranchos, 1800 m. Yahara et al Michoacan, 10km W of Ucareo, along Hwy 15D from Morelia to Maravatio, 2240m. Yahara et al Morelos, On the way from Tres Marias to Mt. Zempoala, m. Yahara & Soejima Morelos, On the way from Tres Marias to Mt. Zempoala, m. Yahara & Soejima Morelos, 7 km S of Tres Marias, on Hwy 95 Cuata from Cuernavaca to Tres Marias, 2620 m. Yahara & 36 3 Soejima 303 Morelos, 5.5 km W from Tres Marias, on the way to Zempoala, 2600m. Yahara et al Oaxaca, 75km S from Puebla-Oaxaca border on Hwy 135 Cuota, 2420m. Yahara & Soejima Oaxaca, 14.6 km NE from JCT in Oaxaca, on the route 175 to Valle National, 2260 m. Yahara & Soejima Oaxaca, 24.8 km NE from JCT in Oaxaca, 3.8 km N of La Cumbre, on the route 175, 2640 m. Yahara & 36 3 Soejima 338 Oaxaca, 40 km SW of Tlaxiaco, on the route 125 to Putla, 2520m. Yahara & Soejima Oaxaca, 33 km SW of Tlaxiaco, on the route 125 to Putla, 2500m. Yahara & Soejima Oaxaca, 5.7km SW of Suchixtlahuaca, 2350m. Yahara et al Oaxaca, 3.3 km W from Ixtran (W edge) to Valle National on Mex 175, 2100 m. Yahara et al Oaxaca, 15.6 km S of Juchatengo, on the way from Puerto Escondido to Oaxaca, 1410 m. Yahara et al Veracruz, 7 km SE from JCT with Route 140 in Perote, on the way to Cofre de Perote, 2800 m. Yahara & 48 4 Soejima 540 Veracruz, 7.2 km SE from JCT with Route 140 in Perote, on the way to Cofre de Perote, 2700 m. Yahara & 36 3 Soejima 826 Veracruz, 18.6km W of Ciudado Mendosa on Hwy 150D, 1980m. Yahara & Soejima Zacatecas, 27.3km from junction to Mex 54, on the way from Jalpa to Tlaltenango, 2370m. Yahara et al Zacatecas, 27.3km from junction to Mex 54, on the way from Jalpa to Tlaltenango, 2370m. Yahara et al Stevia oligophylla Soejima & Yahara Chiapas, 4.8 km NW of Motozintla de Mendoza, on the way to Siltepec, 1610 m. Yahara et al Guerrero, 3.5 km N of JCT to Ixtateopan, on the way from Taxco to Tetipec, 2200 m. Yahara & Soejima B 3 Guerrero, 49 km SW from JCT of Hwy 95 Libre to Filo de Caballo, 2200 m. Yahara & Soejima Guerrero, 8.5 km SW from Filo de Caballo, 63.5 km SW from JCT of Hwy 95 Libre, 2540 m. Yahara & 33 3 Soejima 285 Guerrero, Cerro Alquitran, on the way from Mazatlan to Microwave station, 2330m. Yahara et al ~1B 3 Guerrero, 42 km SW from JCT with Hwy 95, on the way from Chilpancingo to Filo de Caballo, m. Yahara et al. 859 Guerrero, 33.5 km E of Chilapa, on the way from Chilpancingo to Tlapa (Mex 93), 1940 m. Yahara et al Oaxaca, 24.8 km NE from JCT in Oaxaca, 3.8 km N of La Cumbre, on the route 175 to Valle National, m. Yahara & Soejima 339

12 102 A. SOEJIMA ET AL. Appendix I. Continued Species and locality 2n Ploidy level Oaxaca, 34.5 km N from Oaxaca (JCT to 190) to Valle National on Mex 175, 2300 m. Yahara et al Oaxaca, 29.4 km N of Mixtepec, on the way to Oaxaca, 1600 m. Yahara et al Puebla, 14 km NE of Teotitlan on OAX 18 (route to Huautla), 1900 m. Yahara et al Stevia origanoides H.B.K. Durango, 96.6 km NE from Vila Union to Durango on Mex 40, 1920 m. Yahara & Soejima Durango, 96.6 km NE from Vila Union to Durango on Mex 40, 1920 m. Yahara & Soejima Durango, km NE from Vila Union to Durango on Mex 40, 1960 m. Yahara & Soejima ~4B 2 Durango, 42.1 km SW of San Juan de Michis, 4.3 km W from JCT to Las Margaritas, 2505 m, Yahara 44 4 et al. 890 Durango, 28.5 km W of La Ciudad on Mex 40, 2620 m. Yahara et al Durango, 32.5 km W of La Ciudad on Mex 40, 2420 m. Yahara et al Durango, 45.6 km W of La Ciudad on Mex 40, 2290 m. Yahara et al Durango, 118 km NE on Mex 40 from Vila Union, 2280 m. Yahara et al Guerrero, 0.5 km W of Taxco. Yahara et al Guerrero, 5.2 km N of JCT to Ixtateopan, on the way from Taxco to Tetipec, 2300 m. Yahara & Soejima Guerrero, 15.9km E of Tixtra de Guerrero on Route 93, 1800m. Yahara & Soejima Guerrero, 23.7km S of Petaquillas on Hwy 95 Libre, 1100m. Yahara & Soejima ~3B 2 Guerrero, Cerro Alquitlan, on the way from Mazatlan to Microwave station, 2180m. Yahara et al Guerrero, 39km SW from Filo de Caballo, along Chilpancingo-Atoyac route, 2120m. Yahara et al Guerrero, 33.5 km E of Chilapa, on the way from Chilpancingo to Tlapa (Mex 93), 1940 m. Yahara et al Jalisco, 23km NW of Guadalajara on Hwy 15 Cuota, 1580m. Yahara & Soejima Jalisco, 49km W of Ameca, on route 90 to Mascota, 1800m. Yahara & Soejima Jalisco, 18 km S of Talpa de Allende, on the way to La Cuesta, 1440 m. Yahara & Soejima Jalisco, 4 km N of La Cuesta, on the way to Talpa de Allende, 1070 m. Yahara & Soejima Jalisco, 11km E from JCT of Talpa de Allende-La Cuesta route, 2220m. Yahara & Soejima Jalisco, 43km W of Talpa de Allende, on lumber road to Cuale, 1980m. Yahara & Soejima Jalisco, 16km SE of Los Volcanes, on the way to Ayutla, 1970m. Yahara & Soejima Jalisco, 12.5 km S of Autlan on Route 80, 0.5 m S of Puerto del Mazos, 1400 m. Yahara & Soejima Jalisco, 7 8km S of Tecalitlan, along Route 110, 1350m. Yahara & Soejima Jalisco, 7 km NW of San Marcos, E foot of Volcan Colima, 1520 m. Yahara & Soejima Jalisco, 3 km S of San Luis Sayatlan, on a lumber road to N-facing slope, 1950 m. Yahara & Soejima Jalisco, Guadalajara, 11km N on Route 54 from JCT with Periferico, 1300 m. Yahara & Soejima Jalisco, 2.9 km E of El Pueblito, roadside of Mex 15. Yahara & Soejima Jalisco, 8 km W of Guadalajara on Mex 15, 2600 m. Yahara et al Jalisco, 20.5 km SE of junction to route 110, on the way to Jilotran via San Ishidro, 2050 m. Yahara et al Jalisco, 2 km S of San Luis Soyatlan to Cerro Garcia, 1700 m. Yahara et al Jalisco, 2 km S of San Luis Soyatlan to Cerro Garcia, 1700 m. Yahara et al Jalisco, 25.7km N of JCT to Testian on Mex 23, between Guadalajara to Tlaltenango, 1060m. Yahara 33 3 et al Jalisco, 2km E of Villa Guerrero, near a local road from Mex 23 to Villa Guerrero, m. Yahara 33 3 et al Michoacan, 30.2 km E of Morelia on Mex 15, 2130 m. Yahara et al Michoacan, Between Uruapan and Charapendo on R37, 15km S from Uruapan, 1110m. Yahara et al Michoacan, 10km W of Patzcuaro, on the way from Uruapan to Patzcuaro, 2160m. Yahara et al Morelos, On the way from Tilzapotla to El Salto. Yahara et al Morelos, On the way from Tres Marias to Mt. Zempoala, m. Yahara & Soejima Morelos, Tepoztlan, on the way from Tres Marias to Cuernavaca along Hwy 95. Yahara & Soejima Morelos, 14km from Tepoztlan, Tepozteco N.P. Yahara & Ito Nayarit, 4 5km W from La Laguna Santa Maria, on the way to Santa Maria, 960 m. Yahara & Soejima Nayarit, NW slope of Volcan Sanganguey, along small trail to the peak, 1650m. Yahara et al Oaxaca, 120km S from Puebla-Oaxaca border on Hwy 135 Cuota, 1910m. Yahara & Soejima Oaxaca, 11 km NE from JCT in Oaxaca, on the route 175 to Valle National, 1930 m. Yahara & Soejima Oaxaca, 6 km S of Tlaxiaco, on the route 125 to Putla. Yahara & Soejima Oaxaca, 5.7km SW of Suchixtlahuaca, 2350m. Yahara et al Oaxaca, 13.2 km SE from El Camaron to Tehuantepec on Mex 190, 1350 m. Yahara et al Oaxaca, 19.5km N of Juchatengo, on the way from Puerto Escondido to Oaxaca, 1440m. Yahara et al Sinaloa, 43km NE on Mex 24 from Badiraguato, 1180m. Yahara & Soejima

13 DISTRIBUTION AND VARIATION OF STEVIA 103 Appendix I. Continued Species and locality 2n Ploidy level Sinaloa, 57 km NE from Vila Union to Durango on Mex 40, 1020 m. Yahara & Soejima ~2B 3 Sinaloa, 68.1 km NE on Mex 40 from Vila Union, 1200 m. Yahara et al B 3 Veracruz, 19km W of Orizaba on R150 cuota, 1850m. Yahara & Soejima Veracruz, Dos Hermonas, 6km NW of Acajete on Route 140, 2210m. Yahara & Soejima Veracruz, 9 km NW of Acajete on Route 140, on the way to Perote, 2320 m. Yahara & Soejima Veracruz, Dos Hermonas, 6km NW of Acajete on Route 140, 2120m. Yahara & Soejima Veracruz, 13.2km W of Ciudado Mendoza on Hwy 150D, 1780m. Yahara & Soejima Zacatecas, 27.3km from JCT to Mex 54, on the way from Jalpa to Tlaltenango, 2370 m. Yahara et al Stevia ovata Willd. var. ovata Chiapas, 29 km W of San Cristobal de Las Casas, on Hwy 190, 2030m. Yahara et al B 3 Chiapas, 66 km NE of San Cristobal de Las Casas, 18 km NE of Oxchuc, on Hwy 186, 1260 m. Yahara 36 3 et al. 611 Chiapas, 28.8 km NE of JCT 186/190 to Oxchuc on Hwy 186, 2100m. Yahara et al Chiapas, 4.8 km NW of Motozintla de Mendoza, on the way to Siltepec, 1610 m. Yahara et al Chihuahua, 12.8km W from Parral on Mex 24, 1840m. Yahara & Soejima Chihuahua, 12.8km S on Mex 24 from the JCT to Guadalupe y Calvo. Yahara & Soejima Durango, 96.6 km NE from Vila Union to Durango on Mex 40, 1920 m. Yahara & Soejima Durango, 96.6 km NE from Vila Union to Durango on Mex 40, 1920 m. Yahara & Soejima Durango, 96.6 km NE from Vila Union to Durango on Mex 40, 1920 m. Yahara & Soejima Durango, 14.7 km W of Suchil, on the way to San Miguel de la Michilia, 2070m. Yahara et al Durango, 80 km W of Durango City on Mex 40, just W of Llano Grande, 2400m. Yahara et al Guerrero, 14.8 km N of JCT to Ixtateopan, on the way from Taxco to Tetipec, 2080 m. Yahara & Soejima Guerrero, 3.7 km W of JCT to Tetipec, on the way from Taxco to Ixtateopan, 1950 m. Yahara & Soejima Guerrero, 15.9km E of Tixtra de Guerrero on Route 93, 1800m. Yahara & Soejima Guerrero, 15.9km E of Tixtra de Guerrero on Route 93, 1800m. Yahara & Soejima Guerrero, 15.9km E of Tixtra de Guerrero on Route 93, 1800m. Yahara & Soejima Guerrero, 13.8km E of Chilapa on Route 93, 1800m. Yahara & Soejima Guerrero, 49 km SW from JCT of Hwy 95 Libre to Filo de Caballo, 2200 m. Yahara & Soejima Guerrero, 8.5 km SW from Filo de Caballo, 63.5 km SW from JCT of Hwy 95 Libre, 2540 m. Yahara & 60 5 Soejima 281 Guerrero, 8.5 km SW from Filo de Caballo, 63.5 km SW from JCT of Hwy 95 Libre, 2540 m. Yahara & 60 5 Soejima 282 Guerrero, 39 km SW from JCT of Hwy 95 Libre to Filo de Caballo, 2340 m. Yahara & Soejima Guerrero, Cerro Alquitlan, on the way from Mazatlan to Microwave station, 2180 m. Yahara et al ~3B 3 Guerrero, 48.5km SW from Hwy 95, between Chilpancingo to Filo de Caballo, 2050m. Yahara et al Guerrero, 35.8km SW from Hwy 95, on the way from Chilpancingo to Filo de Caballo, 1160m. Yahara 33 3 et al. 873 Guerrero, 16 km E of Tixtla, on the way from Chilpancingo to Tlapa (Mex 93), 1740 m. Yahara et al Guerrero, 13.6 km E of Chilapa, on the way from Chilpancingo to Tlapa (Mex 93), 1800 m. Yahara et al Guerrero, 33.5 km E of Chilapa, on the way from Chilpancingo to Tlapa (Mex 93), 1940 m. Yahara et al Hidalgo, 10km N from Pachuca to El Chico Park. Yahara & Ito, Hidalgo, 10km N from Pachuca to El Chico Park. Yahara & Ito, Hidalgo, 11 km NW from the entrance of El Chico Park. Yahara & Ito, Jalisco, 14km N of Ixtahuacan, 64km N of Guadalajara on route 54, 2040m. Yahara & Soejima Jalisco, 10km N of Ixtahuacan, 60km N of Guadalajara on route 54, 2040m. Yahara & Soejima Jalisco, 49km W of Ameca, on route 90 to Mascota, 1800m. Yahara & Soejima Jalisco, 79 km W of Ameca, 3 km E of Puerto la Campana, on route 90 to Mascota, 1920 m. Yahara & 48 4 Soejima 417 Jalisco, 11km E from JCT of Talpa de Allende-La Cuesta Route, 2200m. Yahara & Soejima Jalisco, 12.5 km S of Autlan on Route 80, 0.5 km S of Puerto del Mazos, 1400 m. Yahara & Soejima B 4 Jalisco, 12.5 km S of Autlan on Route 80, 0.5 km S of Puerto del Mazos, 1400 m. Yahara & Soejima Jalisco, km SE of JCT with Route 110, on the way to Jilotlan via San Ishidro, 1850m. Yahara & 36 3 Soejima 490 Jalisco, 15 km W of JCT with R 110, on the way to Nevado de Colima, 2170 m. Yahara & Soejima Jalisco, 52.8 km S of La Lobera (JCT of Hwy 15D), roadside of Mex 15, 1200 m. Yahara & Soejima Jalisco, 2.9 km E of El Pueblito, roadside of Mex 15. Yahara & Soejima Jalisco, 14.8 km SE of JCT to route 110, on the way to Jilotran via San Ishidro, 1785 m. Yahara et al

14 104 A. SOEJIMA ET AL. Appendix I. Continued Species and locality 2n Ploidy level Jalisco, 14.8 km SE of JCT to route 110, on the way to Jilotran via San Ishidro, 1785 m. Yahara et al Jalisco, 9.5 km SE of junction to route 110, on the way to Jilotran via San Ishidro, 1685 m. Yahara et al Jalisco, 2 km S of San Luis Soyatlan to Cerro Garcia, 1700 m. Yahara et al Mexico, 4.6km E from Mexico-Michoacan border on Mex 15, 2.4km W from Dos Casas, 2580m. Yahara & 24 2 Soejima 819 Mexico, 4.6km E from Mexico-Michoacan border on Mex 15, 2.4km W from Dos Casas, 2580m. Yahara & 33 3 Soejima 820 Mexico, 2km NE on Mex 134 from JCT to Sultepec, between Toluca and Temascaltepec, 3100m. Yahara 36 3 et al. 993 Mexico, 4.9km N on Mex 134 from Tejupilco, 1600 m. Yahara et al Michoacan, 20.3km E of Morelia on Mex 15, 2200m. Yahara & Soejima Michoacan, 20.3km E of Morelia on Mex 15, 2200m. Yahara & Soejima Michoacan, Between Uruapan and Charapendo on Route 37, 15km S from Uruapan, 1110m. Yahara et al Michoacan, 10km W of Patzcuaro, on the way from Uruapan to Patzcuaro, 2160m. Yahara et al Michoacan, 10km W of Patzcuaro, on the way from Uruapan to Patzcuaro, 2160m. Yahara et al Michoacan, 10km W of Ucareo, along Hwy 15D from Morelia to Maravatio, 2240m. Yahara et al ~1B 3 Nayarit, NW slope of Volcan Sanganguey, along small trail to the peak, 1540m. Yahara et al Nuevo Leon, 6.5 km W from Hwy 85, on the way from Santiago to Lagune de Sanchez, 780 m. Yahara & 24, 25, 26 2 Soejima 710 Nuevo Leon, 6.5 km W from Hwy 85, on the way from Santiago to Lagune de Sanchez, 780 m. Yahara & 24, 25 2 Soejima 711 Oaxaca, Conception, 40km S from Puebla-Oaxaca border on Hwy 135 Cuota, 2200m. Yahara & Soejima Oaxaca, 24.8 km NE from JCT in Oaxaca, on the Route 175 to Valle National, 2640 m. Yahara & Soejima Oaxaca, 71 km NE from JCT in Oaxaca, on the route 175 to Valle National, 2600 m. Yahara & Soejima Oaxaca, 1.5 km W of JCT to Numi, 26 km NE of Tlaxiaco, Yahara & Soejima Oaxaca, Microwave station just W of El Tejocote, 18.5 km SW of Huitzo, 2375m. Yahara et al Oaxaca, Microwave station just W of El Tejocote, 18.5 km SW of Huitzo, 2375m. Yahara et al Oaxaca, 4.2 km W from Ixtran (W edge) to Valle National on Mex 175, 2140 m. Yahara et al Oaxaca, 29.4 km N of Mixtepec, on the way to Oaxaca, 1600 m. Yahara et al Puebla, 41.8 km SE of Acatlan on Mex 160, 1800m. Yahara et al Sinaloa, W edge of the village of Huixiopa, 98.4 km NE on Mex 24 from Badiraguato, 880 m. Yahara & 36 3 Soejima 752 Sinaloa, 74.4 km NE from Vila Union to Durango on Mex 40, 1550m. Yahara & Soejima Veracruz, 14km W of Orizaba on R150 cuota, 1680m. Yahara & Soejima Veracruz, 14km W of Orizaba on R150 cuota, 1680m. Yahara & Soejima Veracruz, near State border, on the way from Esperanza to Orizaba on Hwy 150 Cuata, 2560m. Yahara & ca.60 5 Soejima 513 Veracruz, 46km SE from JCT with R140 in Guadalupe Victoria, 3km SE of Ixhuacan, 1660m. Yahara & 33 3 Soejima 545 Veracruz, 46km SE from JCT with R140 in Guadalupe Victoria, 1660m. Yahara & Soejima Veracruz, 13.2km W of Ciudado Mendoza on Hwy 150D, 1780m. Yahara & Soejima Stevia ovata var. expansa Grashoff Nuevo Leon, 36 km S of Monterrey, on the way from Santiago to Laguna de Sanchez, 1480 m. Yahara & 22 +1~2B 2 Soejima 718 Nuevo Leon, 36 km S of Monterrey, on the way from Santiago to Laguna de Sanchez, 1480 m. Yahara & 22 +1~3B 2 Soejima 719 Stevia ovata var. reglensis (Benth.) Grashoff Puebla, 14 km NE of Teotitlan on OAX 18 (route to Huautla), 1900 m. Yahara et al ~1B 3 Stevia ovata var. texana Grashoff USA: Texas, Big Bend National Park, on the way from Basin to Empory Peak, 2000 m. Soejima & 33 3 Mishima 1018 Stevia triflora DC. Chihuahua, 17.8km E of Guadalupe y Calvo, 2500m. Yahara & Soejima ~4B 4 Guerrero, 18 km E of Chilapa on Route 93, 1950 m. Yahara & Soejima

15 DISTRIBUTION AND VARIATION OF STEVIA 105 Appendix I. Continued Species and locality 2n Ploidy level Guerrero, 26km S of Petaquillas on Hwy 95 Libre, between Chilpancingo and Acapulco, 1100m. Yahara & 36 3 Soejima 298 Guerrero, 16 km E of Tixtla, on the way from Chilpancingo to Tlapa (Mex 93), 1740 m. Yahara et al Jalisco, Guadalajara, 11km N on Route 54 from JCT with Periferico, 1300 m. Yahara & Soejima ~2B 4 Jalisco, 4 km SE of JCT with Route 110, on the way to Jilotlan via San Ishidro, 1560m. Yahara & Soejima ~ 2B 2 Jalisco, Guadalajara, 11km N on Route 54 from JCT with Periferico, 1300 m. Yahara & Soejima Jalisco, 4 km SE of JCT to route 110, on the way to Jilotran via San Ishidro, 1450 m. Yahara et al Jalisco, 5.2 km SE of JCT to route 110, on the way to Jilotran via San Ishidro, 1540 m. Yahara et al Jalisco, 5.2 km SE of JCT to route 110, on the way to Jilotran via San Ishidro, 1540 m. Yahara et al Mexico, 4.9km N on Mex 134 from Tejupilco, 1600 m. Yahara et al Michoacan, 10km N of Acahuato, 17km N of Apatzingan on a lumber road, 1250m. Yahara et al Oaxaca, Monte Alvan, 1900m. Yahara & Soejima Oaxaca, 7 km SE of Matatlan, on the way from Oaxaca to Tehuantepec (Hwy 190), 1950 m. Yahara & 36 3 Soejima 371 Oaxaca, 29.4 km N of Mixtepec, on the way to Oaxaca, 1600 m. Yahara et al Oaxaca, 34.8km N of Juchatengo, on the way from Puerto Escondido to Oaxaca, 2100m. Yahara et al Sinaloa, 84.3km NE on Mex 24 from Badiraguato, 680 m. Yahara & Soejima ~3B 3