This file was created by scanning the printed publication. Errors identified by the software have been corrected; however, some errors may remain. CHAPTER 15 Molecular Systematics Daniel L. Nickrent*

Despite thousands of references to dwarf mistle­ isozyme electrophoresis has proven especially valu­ toes, relatively little work has been conducted on the able in providing useful data for examining species systematics or phylogenetics of the genus. Molecular relationships in Arceuthobium. Nickrent and others analyses of intergeneric or interspecific relationships (1984) and Nickrent (1986) first examined the iso­ are even fewer. This chapter reviews previous studies zymes of 19 North American taxa of Arceuthobium. that used macromolecular characters in systematic From that study, it was learned that the genus has studies of Arceuthobium, presents new data, and sum­ remarkably high levels of genetic diversity-67% of the marizes those relationships that appear to be most loci were polymorphic and averaged 2.23 alleles per strongly supported. locus. This result was surprising given the relative homogeneity of the genus with regards to morpholo­ Macromolecular characters that have been exam­ gy and flavonoid composition (chapter 14). ined within the dwarf mistletoes include isozymes and DNA sequences from nuclear ribosomal DNA and a Many of the results of that isozyme study were plastid-encoded gene (rbcL). Using isozyme charac­ consistent with the taxonomic classifications of ters of triploid seed endosperms, interspecific relation­ Hawksworth and Wiens (1972,1984, and table 14.1), ships have been determined for 25 New World including the recognition of 2 subgenera (Arceu­ species, and the results are reviewed below. Subse­ thobium and Vaginata), the close relationship among quent work (Nickrent and Butler 1990, Nickrent and species of section Campylopoda, and the clustering of Stell 1990, Nickrent and Butler 1991) on species com­ A. gillii (sensu lato) andA. vaginatum (sensu lata). plexes within section Campylopoda utilized diploid Isozyme analysis did not, however, support placement shoot tissue, thereby allowing for the use of a wider of A. douglasii and A. pusillum together in section array of population genetics statistics. The genetically Minuta. An unexpected result of that study was the variable internal transcribed spacer (ITS) regions have grouping of A. douglasii, a parasite of Pseudotsuga been sequenced for 22 species of New World and 1 menziesii, with A. divaricatum, a parasite of pinyons. species of Old World dwarf mistletoe (Schuette 1992, That isozyme analysis clearly raised as many questions Schuette and Nickrent 1992, Nickrent and others as it resolved, hence further work was needed, espe­ 1994). Results of analysis of these sequences are com­ cially on Mexican and Central American taxa. pared with those derived from isozymes and other The following portion of this chapter presents characters. Genetically conservative small subunit methodologies and results of an electrophoretic study (18S) ribosomal DNA sequences for 3 species of of 36 dwarf mistletoe populations. The analysis incor­ Arceuthobium and representatives of the other 6 gen­ porates genetic data on 13 populations of7 Mexican era ofViscaceae have been determined. Intergeneric taxa that were not included in the previous isozyme relationships are addressed using these 18S rDNA study (Nickrent 1986) but are pivotal for understand­ sequences and are compared to a similar study ing species and sectional relationships. Some ques­ employing the chloroplast gene rbcL. tions remained after the 1986 study: How is Arceuthobium strictum, the sole species Isozyme Analyses of Interspecific within its series, related to other members of the genus? Relationships Given the discrepancy between Hawksworth and Isozyme electrophoresis has been extensively Wiens (1972) and Nickrent (1986) in sectional used to address questions of population genetics, placement of Arceuthobium divaricatum, what is breeding systems, and systematic relationships in the relationship of A. divaricatum to A. pendens, plants (Soltis and Soltis 1989), Because of the extreme the only other parasite of pinyons? reduction associated with the parasitic habit and the resulting paucity of morphological characters,

·Department of Plant Biology, Southern Illinois University, Carbondale, IL; work supported by grants from the Tinker Foundation, U. S. Department of Agriculture, Forest Service, and National Science Foundation (BSR-8918385). Molecular Systematics 155 Chapter 15

Given their high degree of genetic similarity, are unusually low levels of polymorphism were includ­ members of the Arceuthobiurn gillii complex and ed-Arceuthobium abietis-religiosae, A. globosum the A. vaginatum complex best treated at specific subsp. grandicaule, A. pendens, A. rubrum, A. stric­ or subspecific levels? tum, and A. verticillijlorum. The mean number of alle­ With the inclusion of Arceuthobium abietis­ les per locus for these 6 taxa (5) is lower than the religiosae and A. verticillijlorum in the study, will overall mean for the genus (2.15); this reduction in subgenus Arceuthobium remain monophyletic? allele frequency may be a consequence of their restricted distributions. However, two other Mexican taxa, A. vaginatum subsp. vaginatum and Methodologies A. durangense, show high levels of polymorphism that are comparable to levels found in a related and more Electrophoretic methods are essentially as report­ northern taxon, A. vaginatum subsp. cryptopodum. ed in Nickrent (986). Briefly, seeds are germinated in This contrast in species-to-species variation in poly­ H20 2; viscin is removed from the endosperm; and the morphism agrees with conclusions of Nickrent (986) endosperm is stored at -70°C. Endosperms are then and Hawksworth and Wiens (1972) that section homogenized in an extraction buffer; the buffer Vaginata (which includesA. vaginatum and absorbed onto filter paper wicks; and the wicks insert­ A. durangense) is genetically and morphologically ed into horizontal starch gels. Enzyme separation is more variable than other sections in the genus. effected by electrophoresis, and the gel is sliced and Although A. globosum subsp. grandicaule shows a stained for different enzymes (Wendel and Weeden low level of polymorphism and is also in section 1989). The resulting banding patterns are used to infer Vaginata, only 30 individuals from one population genotypes. Results of gel electrophoresis for 14 indi­ were examined electrophoretic ally, hence this sample viduals of 2 species of Arceuthobium stained for may not be representative of the species' genetic 6-phosphogluconate dehydrogenase appear in figure diversity. 15.1A. Relative intensities of the bands in one lane for heterozygotes ( 4:4: 1 or 1:4:4) confirm the triploid Interpopulational genetic differences were calcu­ nature of the endosperm tissue. In this study, 7 enzyme lated using the chord distance of Cavalli-Sforza and systems were used, representing 9 putative enzyme Edwards (1967). These distances were then used for loci: 6-PGD, IDH, GPI, ADH-1, ADH-2, GDH, UPGMA (Sneath and Soka11973) phenogram con­ G-6-PDH, MDH-2, and MDH-3. struction (fig. 15.2). Values for several combinations are very nearly or equal to 1.0, indicating that few or Genetic interpretation follows that reported in no alleles are shared between the populations being Nickrent (986). Because the genotypes were not compared. These large distance values generally diploid, individual genotypes could not be entered resulted from comparisons alTIOng members of sub­ directly into the statistical program BIOSYS-1 genus Vaginata section Campylopoda and subgenus (Swofford and Selander 1981). Instead, allele frequen­ Arceuthobium. This result is not unexpected because cies were determined for all populations and loci, and subgenus Arceuthobium contains 3 taxa (A. abietis­ these frequencies used as input data. religiosae, A. americanum, and A. verticillijlorum) that Isozyme data for 19 taxa (Nickrent 1986) were are morphologically and genetically distinct from the used in combination with new data for 13 populations rest of the genus. of 7 additional taxa (Arceuthobium abietis-religiosae, A. globosum subsp. grandicaule, A. nigrum, A. pen­ dens, A. strictum, A. vaginatum subsp. vaginatum, and Section Campylopoda A. verticillijlorum), yielding a total of 26 taxa (see the Eleven of the taxa examined are included within appendix "Specimens Examined" for additional data section Campylopoda, series Campylopoda; these taxa on host and collection locality). grouped together at a chord distance of 0.45 or less (fig. 15.2). Two additional taxa of section Campylo­ poda (as delimited in table 14.1) are newly analyzed­ Results and Discussion Arceuthobium pendens and A. strictum. Although they cluster together, the populations are linked at a Genetic variability statistics for the 36 dwarf mistle­ genetic distance value of 054, hence their relationship toe populations appear in table 15.1. The overall mean is distant. The branch comprising these 2 taxa joins the percentage of polymorphic loci is 48.7%, and the mean 11 other taxa of section Campylopoda at the 0.65 level, number of alleles per locus is 2.15. These values are indicating a significant amount of genetic differentia­ lower than those reported in Nickrent (1986) because tion. Arceuthobium strictum and A. pendens are 2 of several populations of Mexican dwarf mistletoes with

156 Molecular Systematics Chapter 15

alleles -a A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 acc acc aaa ccc ccc acc acc aaa acc acc aac bcc aac ccc

alleles -a _b B _c _d _e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 bb ac cc cd ac ce cd aa cc cc ac cc ac be cc de cc ad cd ac cc Figure 15.1-Gel zymograms stained for 6-phosphogluconate dehydrogenase in Arceuthobium. Genotypes are given below each lane number. A Banding patterns from triploid seed endosperms from A. abietinum f. sp. magnificae (No. 1910, lane 1-10) and A. abietinum f. sp. concaloris (No. 1917, lanes 11-14). Note the 1:4:4 or 4:4:1 dosage for heterozygotes. B: Banding patterns for 21 individuals of A. campylopodum (No. 2206) using diploid shoot tissue. Note the 1:2:1 dosage for heterozygotes. the most unusual dwarf mistletoes found in Mexico extremes of this gradient are represented by the "strict" and both have limited distributions: A. strictum is a Campylopoda taxa of the western United States and parasite of Pinus leiophylla and P. teocote and is found the "strict" Vaginata taxa of Mexico. Interestingly, the only in the Sierra Madre Occidentale and in the state of most distant members of series Campylopoda are Durango. Arceuthobium pendens is known only from A. apachecum and A. blumeri; these species occur in Puebla, San Luis Potosi, and Veracruz and like A. divar­ the Southwest and northern Mexico. Whether this icatum parasitizes pinyons. Although Hawksworth observation reflects a "genotypic cline" resulting from and Wiens (1980) placed bothA. pendens and A. divar­ introgression or divergence from a common genotype icatum in section Campylopoda series Campylopoda, is not known. several morphological features and flavonoid chem­ istry (fig. 14.2) gave preliminary evidence that A. pen­ dens was quite distinct from A. divaricatum. This Arceuthobium divaricatum and A. douglasii isozyme evidence is in agreement that these two para­ Arceuthobium douglasii is most closely allied (fig. sites of pinyons are not closely related. 15.2 and Nickrent 1986) with A. divaricatum, not with Placement of Arceuthobium pendens andA. stric­ A. pusillum (section Minuta of Hawksworth and tum in a position intermediate between sections Wiens 1972). This relationship is unexpected because Campylopoda and Vaginata (fig. 15.2) indicates that a the principle hosts of A. douglasii (Pseudotsuga men­ gradation of evolutionary divergence may exist. The ziesix) andA. divaricatum (pinyons) are not closely

Molecular SystematiCS 157 Chapter 15

TABLE 15.1 - Genetic variability at 9 isozyme loci in 36 populations of 26 taxa of Arceuthobium

Mean Mean no. Percent Expected Population Collection sample size of alleles polymorphic Hardy-Weinberg number· number* Arceuthobium per locus per locus locit heterozygosity:!:

1 1917 A. abietinum f. sp. concoloris 42.2 2.0 55.6 0.206 2 1906 A. abietinum f. sp. magnificae 54.7 2.3 44.4 0.183 3 1983 A. abietis-religiosae 34.3 2.0 55.6 0.250 4 2010 A. abietis-religiosae 61.6 1.4 33.3 0.109 5 1932 A. americanum 47.7 3.0 55.6 0.200 6 1929 A. americanum 35.8 3.8 77.8 0.351 7 1945 A. apachecum 61.7 2.0 44.4 0.210 8 1937 A. blumeri 35.1 2.0 22.2 0.133 9 1930 A. californicum 49.6 2.6 66.7 0.253 10 1924 A. campylopodum 28.8 2.1 55.6 0.211 11 1973 A. cyanocarpum 17.6 1.7 44.4 0.124 12 1953 A. divaricatum 80.3 2.7 55.6 0.209 13 1941 A. douglasii 27.7 2.1 44.4 0.228 14 1949 A. douglasii 44.6 2.4 33.3 0.192 15 1870 A. durangense 26.7 2.9 66.7 0.265 16 2049 A. durangense 9.3 2.3 66.7 0.299 17 2051 A. durangense 26.8 3.3 77.8 0.365 18 1938 A. gillii 77.8 2.8 44.4 0.198 19 1996 A. globosum subsp. grandicaule 27.2 1.6 11.1 0.092 20 1801 A.laricis 21.4 1.7 55.6 0.170 21 1947 A. microcarpum 19.1 1.7 33.3 0.154 22 2041 A. nigrum 31.7 2.4 66.7 0.269 23 1962 A. occidentale 37.8 2.0 55.6 0.184 24 1992 A.pendens 20.8 1.8 33.3 0.106 25 1970 A.pusillum 50.9 2.0 22.2 0.103 26 1971 A.pusillum 30.8 1.7 44.4 0.166 27 1853 A. rubrum 8.3 1.3 33.3 0.139 28 2061 A. strictum 24.2 1.1 11.1 0.039 29 1927 A. tsugense subsp. mertensianae 10.0 1.9 66.7 0.290 30 1876 A. vaginatum subsp. cryptopodum 53.0 2.7 44.4 0.172 31 1964 A. vaginatum subsp. cryptopodum 30.2 2.8 55.6 0.256 32 2059 A. vagina tum subsp. vaginatum 8.9 2.0 66.7 0.230 33 1980 A. vaginatum subsp. vaginatum 29.8 2.4 66.7 0.274 34 1981 A. vaginatum subsp. vaginatum 15.4 1.9 66.7 0.233 35 2001 A. verticilliflorum 12.8 1.7 44.4 0.175 36 2065 A. verticilliflorum 4.6 1.3 33.3 0.169

Means 33.3 2.15 48.7 0.200

·See "Specimens Examined" for information on host and locality. t A locus is considered polymorphic if the frequency of the most common allele does not exceed 0.95. *Unbiased estimate (Nei 1978). Direct count heterozygosity not possible given that the allelic data were derived from triploid genotypes.

158 Molecular Systematics Chapter 15 related. These mistletoes are morphologically distinct, Arceuthobium globosum subsp. grandicaule but A. douglasii is so morphologically reduced that emerges as the most distinct member of section association with any taxon is obscure. The concept of Vaginata, as it had from previous morphological and biochemical convergence between these species was physiological studies (Hawksworth and Wiens 1972, discussed by Nickrent (1986) and dismissed in favor of 1984). This taxon has the largest shoot diameter of the a more parsimonious explanation of genetic and phy­ genus (reaching nearly 5 cm), parasitizes at least 12 logenetic similarity. These species share 16 alleles host species, and ranges as far south as Guatemala. across 6 loci, and both species are fixed for GDH66 (an Samples from populations of A. globosum subsp. glo­ isozyme absent in all other species). Both these mistle­ bosum (a more northern subspecies) are needed to toes were included in section Vaginata by Nickrent determine levels of genetic similarity between the 2 (1986); however, addition of other Mexican taxa in the taxa currently treated as suspecies. present analysis suggests that these species more likely The phylogenetic position of Arceuthobium form a transitional group between sections Vaginata rubrum remains somewhat enigmatic because this and Campylopoda. taxon clusters (distantly) with members of section Evidence from flowering groups (Wiens 1968 and Campylopoda based on phenetic analysis of morpho­ chapter 14) also supports the proposed relationship logical data (Hawksworth and Wiens 1972) but the between Arceuthobium douglasii and members of taxon is clearly a component of the Vaginata group section Vaginata. based on isozymes. Arceuthobium rubrum clusters at a genetic distance of 0.6 (fig. 15.2) with A. gillii and Although some species of section Vaginata (e.g., A. nigrum of section Vaginata. In a cladistic analysis A. durangense, A. globosum, and A. vaginatum) have of morphological and physiological features (Nickrent direct spring-flowering, other species of section 1984), A. rubrum was identified as the most basal Vaginata (e.g., A. gillii andA. nigrum) andA. douglasii taxon of the Campylopoda clade; its morphological have indirect spring-flowering. Nickrent (1984) pre­ similarities to section Campylopoda may only be the sents evidence supporting a derivation of indirect result of convergence. Seeds of A. rubrum are ellip­ spring-flowering from direct spring-flowering as a soid and pigmented either green, maroon, or pinto, response to exposure to cooler climates. therefore geographic distribution, isozyme evidence, During these isozyme analyses, thousands of and seed characters support placement of A. rubrum dwarf mistletoe seeds have been examined. Several in section Vaginata. seed features appear to be useful in differentiating Arceuthobium gillii and A. nigrum were originally between sections Campylopoda and Vaginata. Seeds classified as sister subspecies (Hawksworth and Wiens of Vaginata species are often ellipsoid (versa pyriform 1972). In this study, they clustered together at a genet­ to ovoid), have a slight protuberance at the chalazal ic distance of 0.553 and stand apart from the rest of end, have short viscin strands arranged more densely section Vaginata (fig. 15.2). Although only two popu­ near the radicular end, and have endosperm epidermal lations were analyzed, these taxa appear sufficiently cells pigmented with red (or purple/maroon) or varie­ differentiated to justify their elevation to specific sta­ gated green and red (pinto). Because Arceuthobium tus. douglasii seeds are ellipsoid and display the pinto phenotype (A. pusillum seeds are pyriform and entire­ Clustering of taxa related to Arceuthobium vagina­ ly green), the species' affinity to section Vaginata is tum (A. vaginatum subsp. vaginatum, A. vagina tum difficult to ignore. subsp. cryptopodum, andA. durangense) corresponds to the existing taxonomy for some populations (e.g., populations 30,31,32, and 34) but appears genetically Section Vaginata distinct for other populations (e.g., populations 15,16, and 17). The lack of clustering for populations of The new taxa and populations of the predomi­ A. durangense is partly due to the high number of alle­ nantly Mexican section Vaginata included in this study les present at the GPI locus. Population 17 (Puerto are Arceuthobium durangense, A. globosum subsp. Buenos Aires) has 8 alleles at this locus, whereas pop­ grandicaule, A. nigrum, and A. vaginatum subsp. ulation 16 (El Madrofio, only 7 km away) shows just 3 vaginatum. Similarity averaging shows that section alleles at this locus. Although sample sizes for these Vaginata, as defined here, is second only to subgenus populations differ, the results provide evidence for Arceuthobium in terms of within-group heterogeneity significant genetic differentiation over relatively small (fig 15.2). areas.

Molecular Systematics 159 Chapter 15

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 I I I I I I I I I I I I A. abietinum f.s. conc%ris (1 VCc) I A. abietinum f.s. magnificae (2VCc) A. californicum (9VCc) -14 A. /aricis (20VCc) - A. occidentale (23VCc) y A. campy/opodum (1 OVCc) A. cyanocarpum (11VCc) '"" A. microcarpum (21VCc) A. tsugense (29VCc) A. apachecum (7VCc) A. b/umeri (8VCc)

I A. pendens (24VCc) I A. strictum (28VCc) I A. divaricatum (12VCc) I I A. doug/asii (14 VM) - I A. doug/asii (13VM) A. gillii (18VV) A. nigrum (22VV) rr A. rub rum (27VCr) I A. vaginatum subsp. cryptopodum (30VV) I A. vaginatum subsp. cryptopodum (31 VV) - rl A. durangense (1SVV) ~ - A. durangense (17VV) A. vagina tum subsp. vagina tum (32VV) '"' - Lfi A. vaginatum subsp. vagina tum (34VV) A. vagina tum subsp. vagina tum (33VV) A. durangense (16VV) A. g/obosum subsp. grandicau/e (19VV)

I A. pusillum (2SVM) I A. pusillum (26VM)

J A. americanum (6A) I ,. A. americanum (SA) I A. verticilliflorum (3SA) I - A. verticilliflorum (36A) I A. abietis-re/igiosae (3A) I A. abietis-religiosae (4A)

1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Chord Distance (Cavalli-Sforza & Edwards 1967)

Figure 15.2 -The UPGMA phenogram resulting from isozyme analysis of 36 populations of Arceuthobium. Cophenetic correlation = 0.968, stan­ dard deviation 6.6%. Codes following species name indicate population number (table 15.1) and taxonomic affilation according to the classifica­ tion of Hawksworth and Wiens (table 14.1 compare with table 15.2). Key: A = subgenus Arceuthobium, V = subgenus Vaginata, V = section Vaginata, C = section Campylopoda, M = section Minuta, c = series Campylopoda, r = series Rubra, s = series Stricta.

160 Molecular Systematics Chapter 15

Arceuthobium pusillum appears, however, that the majority of section As reported in Nickrent (1986), isozyme evidence Campylopoda species are found in the United States, indicates that Arceuthobium pusillum is a distinct where as section Vaginata species predominate in taxon in the genus that is placed midway between sub­ Mexico. genus Vaginata and Arceuthobium. Isozyme analysis The two parasites of pinyons, Arceuthobium does not support a section Minuta composed of A. divaricatum and A. pendens, are not closely related. pusillum and A. douglasii. These results agree with This conclusion is supported by the isozyme data, those of Crawford and Hawksworth (1979 and see markedly different flavonoid chemistry, systemic table 14.2), which show that these two species have broom formation in A. pendens, and different hosts different flavonoid patterns. Isozyme data also do not (Hawksworth and Wiens 1980). provide evidence that A. pusillum and A. douglasii are recent derivatives from section Campylopoda as sug­ Arceuthobium gillii and A. nigrum are clearly gested by Hawksworth and Wiens (1972, page 38). related but cluster at a genetic distance value of 0.553, That these species share isophasic broom develop­ thus supporting their recognition as distinct species. ment and indirect spring-flowering must be interpret­ The relatives of A. vaginatum have high levels of ed as convergence. genetic diversity, and cluster analysis indicates a sub­ stantial genetic differentiation between populations. Further work is required to better understand appor­ tionment of genetic variation in the Arceuthobium Subgenus Arceuthobium vaginata complex. Only Arceuthobium americanum represented subgenus Arceuthobium in the earlier isozyme study Subgenus Arceuthobium shows greater within­ (Nickrent 1986). With the inclusion of A. abietis­ group heterogeneity than any of the other groups. religiosae andA. verticillijlorum, all three New World Despite the variation, isozyme analysis places members of the subgenus have been examined elec­ Arceuthobium abietis-religiosae, A. americanum, and trophoretically. These species share several isozymes A. verticillijlorum on a single branch that joins the (IDH136, GPI200, G-6-PGDI00, and MDH-3187) and remainder of the species at a genetic distance of 0.82. occupy the same branch of the phenogram (fig. 15.2). Thus, isozyme data support use of verticillate sec­ These results demonstrate that they are at least distant­ ondary branching (Mark and Hawksworth 1981) as a ly related. A comparison of genetic distance values distinguishing character for the subgenus. indicate that A. americanum is more closely related to A. verticillijlorum than it is to A. abietis-religiosae. The two populations of A. abietis-religiosae showed a Species Relationships in the Arceuthobium genetic distance of 0.452; this relatively high value campylopodum Complex indicates that substantial genetic differentiation has Additional isozyme analyses have been conducted occurred between populations separated by only on various members of the Arceuthobium campylopo­ 400 km. This genetic differentiation is further demon­ dum complex (Nickrent and Butler 1990, Nickrent and strated by the fixation for different alleles at the IDH Stell 1990, Nickrent and Butler 1991). These studies locus (IDH136 and IDH160). According to similarity/ utilized diploid shoot tissue as a source of isozymes, distance measure averaging, subgenus Arceuthobium hence a larger number of population genetic analyses is the most heterogeneous of all the dwarf mistletoe could be conducted. Enzyme banding patterns typical groups examined, andA. abietis-religiosae occupies of diploid tissue are shown in fig. 15.1B. Based upon the most basal position among the species examined. UPGMA cluster analysis, California coastal populations of Arceuthobium parasitic on Pinus radiata and P. muricata are genetically distinct from A. campylo­ Conclusions podum (sensu stricto) and are segregated at the specif­ With the new isozyme evidence, questions raised ic level as A. littorum. Examination of isozymes from in the introduction can now be answered. Arceutho­ allopatric and sympatric popUlations of A. campylo­ bium strictum is distantly related to most other taxa in podum and A. occidentale did not reveal significant section Campylopoda and clusters with A. rubrum. genetic differentiation, hence they could be consid­ These relationships are not totally in conflict with the ered a single biological species. classifications of Hawksworth and Wiens (1972 and Nickrent and Butler (1991) examined genetic rela­ table 14.1), in which these species were placed in their tionships among dwarf mistletoes related to own series (Stricta and Rubra, respectively). It Arceut~obium campylopodum that are parasitic on

Molecular Systematics 161 Chapter 15

Pinus attenuata and P monticola of northwestern via unequal crossing over and other DNA turnover California and southwestern Oregon. The parasites of mechanisms. This phenomenon is referred to as "hori­ P attenuata and P monticola were genetically distinct zontal" or "concerted evolution" (Brown and others from each other. These species were named, respec­ 1972, Arnheim and others 1980, Arnheim 1983). tively, A. siskiyouense and A. monticola (Hawksworth Within the cistron, ribosomal genes and spacers show and others 1992b). This result is in accordance with markedly different tempos of evolution Oorgansen the observation that the Klamath-Siskiyou Mountain and Cluster 1988), hence studies of closely related and region contains a highly diverse and endemic flora. distantly related organisms can be conducted using the These dwarf mistletoes likely represent the most appropriate region. The ITS regions and enclosed 5.8S recent evolutionary lines to diverge from the A. campy­ rDNA were sequenced and analyzed in 22 species of lopodum complex. Arceuthobium to allow a comparison with phylo­ genies generated from other methods. The results from electrophoretical examination of the three host-forms of Arceuthobium tsugense (mountain hemlock, western hemlock, and shore pine) indicate that the population infecting Tsuga Methodologies mertensiana is genetically distinct and deserving of Sequence analyses were conducted on samples taxonomic recognition as A. tsugense subsp. merten­ from 24 populations of Arceuthobium that represented sianae (Nickrent and Stell 1990, Hawksworth and oth­ 22 taxa (see fifth appendix, "Specimens Examined"). ers 1992b). Arceuthobium tsugense subsp. tsugense Both shoot and seed materials were used as sources consists of two morphologically similar host races par­ for DNA. Genomic DNA was obtained from shoots by asitic on Tsuga heterophylla (western hemlock race) grinding in liquid nitrogen and extracting in 2x CT AB and Pinus contorta var. contorta (shore pine race). (Doyle and Doyle 1987). Crude homogenates of seeds Isozyme analysis failed to detect significant genetic dif­ were made in a buffered protease solution (Schuette ferentiation between these two host-forms, hence 1992, Nickrent and others 1994). they were retained within the same subspecies. The polymerase chain reaction (PCR) was used to amplify the ITS region from the genomic DNA extract (Mullis and Faloona 1987). A number of conserved Species Relationships From sites on both the 18S and 26S rDNA allow the construc­ Ribosomal DNA Spacer Sequences tion of forward and reverse primers that bracket ITS-I, 5.8S rDNA, and ITS-2. A common primer combination Many studies addressing interspecific relationships employed the 18S 1830 forward and 26s 25 reverse in plants have been conducted using restriction site (5" AACAAGGTTTCCGT AGGTGA -3" and 5" TATGCT­ data from chloroplast DNA (see Soltis and others TAAAYTCAGCGGGT-3" respectively), which yielded 1992). Few studies have used DNA sequences to a 0.64-kb fragment upon symmetrical amplification. examine interspecific relationships because a gene or This fragment was used as a template for an asymmet­ segment of DNA that is of adequate size and that rical PCR reaction (Gyllensten and Erlich 1988) that evolves at a sufficiently fast rate is required. Recently, produces single-stranded DNA. In some cases, the the internal transcribed spacer (ITS) regions of the double-stranded template was sequenced directly. ribosomal DNA cistron have been shown to evolve at Because Arceuthobium abietis-religiosae andA. rates appropriate for examining more recently diverg­ oxycedri apparently had mutations at one or both of ing lineages (Baldwin 1993). As shown in fig. 15.3, the the above two priming sites, alternate internal18S (for­ rDNA cistron (transcriptional unit) comprises (from 5" ward) and 26S (reverse) primers were used. The PCR to 3") an external transcribed spacer (ETS), the 18S product was then gel purified for subsequent sequenc­ rDNA, ITS-I, 5.8S rDNA, ITS-2, 26S rDNA, and a tran­ ing reactions (Nickrent 1994). These sequencing reac­ scription termination site (TIS). This cistron is tions were carried out using the terminal amplification tandemly repeated several hundred to several thou­ primers or forward and reverse primers constructed sand times in the nucleolar organizing region for the conserved sites on the 5.8S rDNA (sites 32 to (Arnheim 1983). 52). Chain-termination sequencing reactions using The transcriptional units themselves are separated dideoxynucleotides (Sanger and others 1977) were by a nontranscribed spacer (NTS). The NTS region has conducted using Sequenase® (U.S. Biochemical Corp.). been used to study variation at the population or even An autoradiogram with ITS sequences for three individual level (May and Appels 1987). Although dwarf mistletoe species is shown in figure 15.4. Most ribosomal cistrons occur in high copy number, the mutations were base substitutions, thus allowing man- sequence of each repeat within a genome is conserved

162 Molecular Systematics Chapter 15

11 kb I I ~====~------LZDn LLZL~···· ------

1.0 kb Figure 15.3 -The organization of higher plant ribosomal DNA cistron. NTS = nontranscribed spacer, ETS = external transcribed spacer, 18S = small-subunit rDNA, ITS-l = first internal transcribed spacer, 5.8S = rDNA, ITS-2 = second internal transcribed spacer, 26s = large-subunit rDNA, TIS = transcription termination site. The rDNA cistron is tandemly repeated many times within the plant genome. ual alignment using "Eyeball Sequence Editor" (Cabot ITS sites can be classified according to their variability: and Beckenbach 1989). Minimum length trees and 39% are mono typic, 33% are ditypic, 15% are tritypic, other analyses of the aligned sequences were conduct­ 2% are tetratypic, and 11 % are insertions or deletions. ed using PAUP version 3.1 (Swofford 1993). These values were determined without the inclusion of A. abietis-religiosae and A. oxycedri because of their high sequence divergence compared with other taxa Results and Discussion (see below). In Arceuthobium, ITS-l is less variable than ITS-2 and is also smaller (which is the case for Overall, little length variation occurs. ITS-l many other, mainly asterid, species). At 208 bp, dwarf sequences varied by less than 1% from the average mistletoe ITS-l is within the range (194 to 265 bp) length (208 base pairs); ITS-2 varied by less than 1.5% reported for other angiosperms (Baldwin 1993). In when compared to the average (226 bp). The 5.8S Calycadenia and other composites, as well as Vicia rONA sequences were usually 167 bp in length, and Sinapis, ITS-l is larger than ITS-2 (Baldwin 1993). although the three members of subgenus Arceutho­ bium and Arceuthobium divaricatum had a 5.8S rONA The alignment ofITS-l, 5.8S rONA, and ITS-2 pro­ of length 166 bp. This is slightly larger than the com­ duced a matrixof22 Arceuthobium species by 619 mon length (164 bp) seen in other angiosperms. The sites (a portion is shown in fig . 15.5). Of these sites, 388 (62.6%) were variable. Because A. abietis­ ABC 0 E F religiosae and A. oxycedri had high sequence diver­ ACGT ACGT ACGT ACGT ACGT ACGT gence compared with other taxa and could not be aligned beyond bp 470, these species are excluded from further variability calculations. Among the remaining 20 taxa, 304 variable sites were present. Of these, 174 were phylogenetically informative (Le., a different nucleotide shared by at least 2 taxa). ITS-l contained 71 sites (40.8% of total); 5.8S had 15 sites (8.6%); and ITS-2 had 88 sites (50.6%). When the num­ ber of phylogenetically informative sites is taken as a percentage of the number of variable positions, the following values were obtained: 53.7% (71/132) for ITS-I , 46.8% (15/ 32) for 5.8S, and 63.7% (88/138) for ITS-2. To test for possible significant intraspecific varia­ tion, partial ITS sequences (100 bp) were determined for individuals from 3 populations of Arceuthobium americanum and 2 populations of A. diva rica tum. A Figure 154 -Autoradiogram showing ITS sequences for three single change was detected in A. americanum, and no Arceuthobium species. Left set for each species utilized the 26s 25 differences were seen in A. divaricatum. Several taxa reverse primer, right set the 18S 1830 forward. A and B: A. apach· have been sequenced twice from either the same ecum (No. 1945). C and D: A. pusillum (No. 1969), E and F: genomic DNA sample or a second crude seed extract. A. vaginatum subsp. crytopodum (no. 1978).

Molecular Systematics 163 Chapter 15

oX'{ GUA ...• D •• G .••. G.• G••.•••• D •. C •••.•• AUAAA .• W ••• G ••• D ••• CU.-. Section Campylopoda ABR .-A ..•. D •• G ••.• G •• G •.••.•• G .• C •••••• AUAAA •• W ••• G ••• D ••• C •• D. AME DCGUGCUcUUAAAGADGAAUGACAMUAAUAAGWACACU-ADCCUAGADCGADGADCUAG 60 As evidenced from the clustering and branch VER ••.•••. D.A •. G••.••.•••••• G.C.C •••.•• A ••••••••.••••• UC •••••••• PEN •• A •••. D.C ••••••.•••.•.•• G••• C.D •• GU.C .•••. W.D •••• AD ••••••• D lengths, four members of section Campylopoda are GUA •• A ..•• D.C •••••••••••••.• G ••• C.D •• GUAC ••••• W ••••.• AD ••••••. D genetically very similar-A. abietinum f. sp. magnifi­ PDS .• A.A •. D •••.••.•••• CC ••. DGC .•••••••• AG ••• G.W •••••. CU ••••• C.A BIC .• A •••. D ••••••••.• C-••.• DGC ••.• - •••• AG ••• G.W ••••••• D ••••• C.A cae, A. apachecum, A. campylopodum, andA. micro­ GID •• A.A .• D •••••••••.•••••.• GC .• C •••••• AG •••.• W ••••••• D ••••••• A OIV ••.•••• UK .•••..•.• G .•••.• GC •• A ••••.• AGAC ••• W .•••••• C •••••••• carpum. Sequences of A. cyanocarpum, A. occiden­ IXlU •• A •••• D ••••••••••••••••• GC .• A •••••• AG ••••. W ••••••• D ••••••• A tale, andA. tsugense were either very similar or identi­ GIG •• A •••• D ••••••••••••••••• GC ..••••••• AG •••• CW ••••••• D •••••. -A NIG .• A ••. CU ••••' ••••••.•••••• GC .••. - •••• AG ••••• W .•••••• D ••••••• A cal to those of the above four taxa and therefore not RUB •• A •••• D ..•.•••••• D •..••. GC ••••••• G.AG ••••. W ••• G ••• D •••••• -A included. The section Campylopoda clade, compara­ VAC •• A ••• CU ••. G••••••••••••• GC •••••.••• AG ••.•• W ••••••• D ••••••• A VAV •• A •..• D ••••••.•.•••••••• GC ••••••••• AG ••••• W ••••• D.D ••••••• A ble to series Campylopoda (as delimited by OUR .• A •••• D •.•.•••••••••...• GC •.••••••• AG ••••• W ••••••• D ••••••• A STR •• A •••• D•••••••••••.•.••• GC •.• DG ••• GAG ••.•• W •••.••• D ••••••• A Hawksworth and Wiens 1972, 1984, and table 14.1), ABM •• A.R •• D ••••••.•.••• R ••.• G•.• AG ••.•• AD ••••• MD .•••••• D ••••••• A occurs mainly in the United States and does not APA .• A •••• D •••.••••..•••.•.• G••• AG ••••• AD •••.• W ••••••• D ••••••• A CAM •• A •••• D ••••.••••••••.••• G••• AG •••.• AD ••••• W ••••••• D •.•••••A include Mexican and Caribbean species such as A. MIC •. A •.•• D •..•.••••••.•.••• G••. AG •.••• AD •.••• W ••••••• D ••••• , •• A guatemalense, A. pendens, A. rubrum, A. bicarinatum, OYX ••• - •• D •• GCG .• W.G •• W.CA.GCC.C ••• DG.CA •• A.AD.D.UA.C •••• C.G.K and A. strictum. The latter three of these species were ABR ••• - •• D •. GCG •• W.G •• W.CA.GCC.C ••• DG.CA •• A.AD.D.UA.C •••• C.G.D AME VAGGACAUWGACACAWGUCAGGGAAD-ADGUGCUCUCWGWCCAMUAUAAAWGACA 120 previously segregated into series Rubra (A. rubrum VER .G ••••••••• G •••.••••••• D.• - ••••••• D •• G•••.•• D.C.VA •••••••• G .• and A. bicarinatum) and Stricta (A. strictum) PEN CG. DG .••. C •• D •••• G.CG •• C •.••••••• ADGAGU ••••• D ••• D •• C •• D •••••• GUA C •• AC •••• C .• D••••••• G •• CA •• G .•••• ADGUGUG •••. D •• W ••••••• C.G •• (Hawksworth and Wiens 1972 and table 14.1), thus PDS .•• A ••.•• CAD .••• CGA. DG. D .•• C •••••• DGUGU •••.• W •• D ••• G ••• C.GU. BIC .D.A ••••• CAD .••. CGA.DG.C .•• C •••••• DGUGU ••••• W •• D ••• G ••• C.GU. providing some indication of their differentiation from GID ••• A ••••• C .• D••••••• DG.D .•• C .•.••• DG--D ••••• W •• D ••• G ••• C.GU. series Campylopoda. ITS sequence analysis does not OIV .G.A ••••• C ••.••••••• DG.D .•• C •••.••• GUGU •••.• D ••• D ••• G •• Cc.GU. IXlU ••• A ••••• CU ••••.• G •• DG.C ••• C .••••• DGUGU ••••• W •• D ••• G ••• C.GU. support a close relationship among members of these GIG •• VA •.•• GC ••••.•• G •• DG.C ••. C •.•••• DGUGU ••••• DG •• D ••• G.D.C.GU. three series; but rather, the analysis indicates various NIG •.• A .•••• C .•.•••• G•• DG.C .•• C •••••• DG.GU •••.• W •• D ••• G••• C.GU. RUB •• M. Y••• C •• D .••• G •. DG. C ••• C .••••.• GAGU •.••• W •• VA •• G. D.C.GU. relationships to members of section Vaginata. VAC ••• A •.•.• C .••••.• G•. DG.C .•• C .•••.• DG.GU ••••. VA •• D ••• G ••• C.GU. VAV •.• A •••.• C ••••••• G•• DG.C .•••.• C •.• DG.GU •••.• W •• D ••. G ••• C.GU. A strongly supported result of the ITS analysis OUR ••• A ••••• C ••..••• G .. DG.C ••• C .••••• DG.GU ••••• W •. D ••• G ••• C.GU. STR .• A •••••. C ••••• G.G •• DG.C ••. C •.•••• DG.GU ••••• W •• D ••• G ••• C.GU. (100% bootstrap) is the association of Arceuthobium ABM •.• A ••••. C .• D •••• G •• DG.C ••• C ••••.• DG.G •••••• W •• D ••• G ••• C.GU. APA ••• A .•••• C •• D•••• G •• UG.C .•• C ••••.• DG.G •••••• W •• D ••• G ••• C.GU. guatemalense with A. pendens. This clade appears CAM ••• A ••.•• C •• D •••• G•• DG.C ••• C .••.•• DG.G •••••• W •• D ••• G ••• C.GU. basal to all other members of subgenus Vaginata, not MIC ., .A ••••• C .• D•••• G•• DG.C ••• C •.•••• DG.G •••••• W •• D ••• G ••• C.GU. as a component of section Campylopoda, series Figure 15.5 -Sequence alignment showing the first 120 bp of ITS-1 Campylopoda (as delimited by Hawksworth and for 22 species of Arceuthobium. A dot indicates the same base as the Wiens 1984 and table 14.1). Arceuthobium reference CA. americanum). Ambiguous nucleotide designations fol­ guatemalense is confined to the mountains of low IUPAC specifications Ce.g., Y = C and 1). Key: OXY = A. oxycedri, ABR = A. abietis-religiosae, AME = A. americanum, VER = A. verticil­ Guatemala and southern Mexico, where it parasitizes lijlorum, PEN = A. pendens, GUA = A. guatemalense, PUS = A. pusil­ Pinus ayacahuite (of subgenus Haploxylon). lum, DIV = A. diva rica tum, DOU = A. douglasii, GIG = A. gil/ii, NIG = A rceuthobium pendens is known only from Puebla, A. nigrum, RUB = A. rubrum, VAC = A. vaginatum subsp. cryptopo­ San Luis Potosi, and Veracruz, Mexico, and is parasitic dum, VA V = A. vaginatum subsp. vagina tum, DUR = A. durangense, STR = A. strictum, ABM = A. abietinum f. sp magnificae, APA = A. on the Haploxylon pines, P. discolor and P. orizaben­ apachecum, CAM = A. campylopodum, MIt = A. microcarpum. sis. Both of these mistletoes and their hosts are narrow endemics. Given their position on the phylogram and Only very rarely were true polymorphisms detected, their endemic distributions, these species could repre­ and these were subsequently coded in the matrix as sent relictual taxa that diverged early during the migra­ ambiguous nucleotides. tion and evolution of Arceuthobium in the New World. The data matrix of 22 taxa by 619 sites produced 6 equally parsimonious trees of 735 steps. Bootstrap analysis with 200 replications was conducted to test Arceuthobium divaricatum andA. douglasii the reliability of the resulting clades (fig. 15.6). As Although classified into different sections by expected from their large sequence divergence, Hawksworth and Wiens (1972 and table 14.1), Arceuthobium abietis-religiosae and A. oxycedri form a Arceuthobium divaricatum and A. douglasii cluster clade well removed from the remainder of the genus. together according to isozyme analysis (Nickrent Removing these species from the analysis and substi­ 1986). Arceuthobium divaricatum occurs near the tutingA. americanum andA. verticillijlorum as out­ base of section Vaginata and near A. globosum in the groups resulted in the same topologies. Specific rela­ derived phylogram (fig. 15.6). When ITS-1 alone is tionships derived from this analysis are discussed analyzed (tree not shown), A. divaricatum and A. below. douglasii cluster together with a high bootstrap confi­ dence. Examipation of the alignment shows that

164 Molecular Systematics Chapter 15

4 A.oxycedri A. abietis-religiosae 22 31 A. verticilliflorum 100 ...... ______28 A. americanum

36 21 A. guatemalense 100 24 A. pendens 198 20 19 A. pusillum 28 « 100 L...-______A. bicarinatum 4 5 2 A. durangense 98 4 A. vaginatum ssp. cryptopodum ...... __2_4 ___ A. vaginatum ssp. vaginatum '--__2_s ___ A. strictum 24 7 100 A. nigrum 12 8 1...-__ A. gillii ----2-6---A.rubrum 18 r------A. douglasii 2 3 A. abietinum f. Sp. magnificae 6 37 1 A. apachecum 100 4 A. microcarpum 17 87 1 100 A. campylopodum 30 a....-______A. divaricatum 12 1...-__ A. globosum Figure 15.6 -One of 6 equally parsimonious trees oflength 738 derived from analysis of Arceuthobium ITS-I, -2, and 5.8S rDNA sequences. The numbers above the branches indicate number of nucleotide subsitutions and the numbers below the branches indicate percentage values from 200 replications derived from the bootstrap majority rule consensus tree. Clades with no bootstrap value indicated were not strongly supported (present in <50% of the trees). Consistency index = 0.755, homoplasy index = 0.286, retention index = 0.726.

A. divaricatum has a divergent ITS-2 sequence com­ surprising, although isozyme analysis had shown pared to other members of subgenus Vaginata. genetic divergence between it and species in series Taking the isozyme and ITS evidence together, a clade Campylopoda. All four of these taxa parasitize pines of composed of A. divaricatum and A. douglasii is subgenus Diploxylon and their distributions range presently favored. from the northern Sierra Madre Occidental (Durango through Chihuahua and Sonora) to the southwestern Section Vaginata United States (Arizona, New Mexico). Two strongly supported clades representing sec­ The second clade is composed of Arceuthobium tion Vaginata are seen following bootstrap analysis rubrum, A. gillii, and its recent segregate species, A. (fig. 15.6). The first is composed of Arceuthobium nigrum. The association of A. gillii with A. nigrum is vaginatum subsp. vaginatum, A. vaginatum subsp. strongly supported by ITS analysis (96% bootstrap). cryptopodum, A. durangense, and A. strictum. Previous classifications (Hawksworth and Wiens 1972) Bootstrap support is 75% for the entire clade contain­ ing A. rubrum. Because isozyme characters also indi­ and isozyme studies (Nickrent 1986 and this chapter) have shown relationships among the first three of cated a grouping of these three species, their phyletic affinity is highly probable. these taxa. The addition of A. strictum is somewhat

Molecular Systematics 165 Chapter 15

Arceuthobium pusillum and A. bicarinatum This route to Hispaniola via Central America was pro­ A surprising but strongly supported clade (100% posed by Rosen (1975) as a "vicariance" pathway. But bootstrap) contains Arceuthobium pusillum and given the molecular evidence, entry into Hispaniola A. bicarinatum. The former species is a reduced para­ via eastern North America and Cuba is favored over site of spruce of the northern United States and the southwest track via Honduras. Canada, and the latter is a relatively large parasite of The present lack of parasitism of low-elevation Pinus occidentalis on the island of Hispaniola. pines in Honduras, Belize, Cuba, Hispaniola, and the Hawksworth and Wiens (1972) placedA.pusillum and southeast United States indicates that an ancestral A. douglasii in section Minuta, a placement not sup­ species was either already adapted to high-elevation ported by isozyme analysis (Nickrent 1986). They also hosts or that, subsequent to speciation, the low-eleva­ suggested thatA. bicarinatum arrived in Hispaniola tion parasites became extinct. Even if Arceuthobium via a Central American land bridge that was connected bicarinatum were found to be genetically similar to to Honduras during the late Tertiary Period (see chap­ A. hondurense, this relationship would not favor one ter 5). This route seemed plausible given the morpho­ migration track over the other. These species would logical similarity between A. bicarlnatum and A. hon­ simply be a relictual taxon derived from the originally durense. The distance from Hispaniola to the nearest widespread ancestor. Given the overall climatic deteri­ extant population of A. pusillum is about 2,300 km; A. oration and the accompanying extinctions that bicarinatum and A. hondurense are presently separat­ occurred in eastern North America during the ed by about 1,100 km. Oligocene Epoch (Tiffney 1985), this ancestral mistle­ Given these biogeographical distributions and the toe is likely now extinct. The isolated position of A. DNA sequence results, an alternate hypothesis regard­ bicarinatum on high-elevation pines of Hispaniola ing these two mistletoes is suggested. Several plant suggests that it is indeed a Tertiary relict. species found at high elevations in Hispaniola are Morphological differences between Arceutho­ known to have close relatives in the eastern United bium pusillum and A. bicarinatum contrast with their States, such as Lyonia 0udd 1981) andJuniperus high level of genetic similarity. High genetic variability (Adams 1989). Hawksworth and Wiens (1972) sug­ as measured by isozymes and increased substitution gested that Arceuthobium arrived in the New World in rate at the nuclear ribosomal cistron suggests that A. the early Tertiary Period via a Beringian land bridge. If pusillum is more genotypically variable than is out­ one accepts that the late Tertiary microthermal vegeta­ wardly apparent from its morphology. Sufficient diver­ tion was largely derived from the preceding flora of sity of pathogenicity genes apparently exists to allow that area (Wolfe 1975), then the ancestor to A. bicari­ this species to parasitize Larix laricina, Pinus strobus, natum and A. pusillum was likely already present in P. resinosa, and P. banksiana, in addition to the eastern North America in the early Tertiary Period. spruces Picea mariana, P. glauca, and P. rubens, its Arceuthobium pollen (likely A. pusillu m ) is known three principal hosts. It is hypothesized that A. pusil­ from as far south as Georgia from the Pleistocene lum and A. bicarinatum represent morphologically Epoch (Watts 1975 and table 5.10), hence dwarf mistle­ divergent endpoints of lineages that have been shaped toes have occupied the eastern United States in past by quite different evolutionary forces. geological times. Evidence that some Arceuthobium species such as A. oxycedri were already well differen­ The above genetic data on the modern species tiated by the Miocene Epoch (Stuchlik 1964) also sug­ suggests that their ancestor likely possessed a large gests evolution of the genus in the early or mid­ store of potential genetic variation that became mani­ Tertiary Period. fest following diversifying selection. Increased fitness was attained by separate populations exploiting differ­ Configuration of the Caribbean region with ent environments and hosts. The reduction in shoot respect to North and Central America during the height, systemic broom formation, spring flowering, Tertiary Period is an area of active research. In the later and rapid fruit maturation seen in Arceuthobium pusil­ Paleocene to mid-Eocene Epochs, the Greater Antilles lum may represent adaptations to greater winter collided with the Bahama Platform (Pindell and Barrett extremes, as occurred in other eastern North American 1990) and connected Cuba to the North American plant species. plate. By the Miocene Epoch, Hispaniola was contigu­ ous with the eastern part of Cuba, and northeastward displacement was occurring along the Oriental Fault. Subgenus Arceuthobium The connection between Honduras, Jamaica, and the Greater Antilles via the Nicaraguan Rise was likely sev­ Two of the most striking results of this study of ITS ered during the middle Cenozoic Era by subsidence. variation are t~e extreme divergence of Arceuthobium

166 Molecular Systematics Chapter 15 abietis-religiosae and A. oxycedri from the other taxa mined by Morgan and Soltis (1993). Discussion of sampled and the similarity of these species to each these analyses provides insight into the phylogenetic other. These results require a modification of con­ position of Arceuthobium within the family. cepts regarding relationships among the three New World members of subgenus Arceuthobium as well as their relationship to Old World members of this sub­ 18S rDNA and rbcL Sequence genus. Analysis Arceuthobium americanum and A. verticillijlorum are more closely related to each other than to any The 18S rDNA sequences are approximately 1,805 other species, as shown by 100% bootstrap confidence bp in length and can be aligned manually given infor­ for their clade. This clade has more affinity with sub­ mation on secondary structural features (Neefs and genus Vaginata than with A. abietis-religiosae and others 1990, Nickrent and Sargent 1991). On average, A. oxycedri-indicating a major divergence in the ver­ 1 to 5% of the sites are variable when comparisons are ticillately branched group during their evolution in the made among most flowering plants; higher rates of New World. Subgenus Vaginata was apparently nucleotide substitution are observed in the parasitic derived from an ancestor shared with A. americanum angiosperm families Rafflesiaceae, Hydnoraceae, and A. verticillijlorum. The extreme divergence of Balanophoraceae, and Viscaceae (Nickrent and Starr A. abietis-religiosae andA. oxycedri from the remain­ 1994). Given this higher evolutionary rate, parsimony ing species also could be interpreted as evidence of analysiS using PAUP (Swofford 1993) was employed to separate migrations into the New World. Further mol­ study relationships among Arceuthobium oxycedri, A. ecular work would greatly benefit from inclusion of pendens, A. verticillijlorum, Dendrophthora clavata, additional Old World species, such as A. azoricum, D. domingensis, Ginalloa arnotiana, Korthalsella A. chinense, A. juniperiprocerae, A. minutissimum, A. complanata, K lindsayii, Notothixos leiophyllus, N pini, and A. tibetense. subaureus, Phoradendron californicum, P. serot­ inum, Viscum album, and V articulatum (see "Species Examined" for host and collection data). In addition, the sequence of Antidaphne visco idea (Eremolepid­ Intergeneric Relationships in aceae) and Santalum album were used as outgroups. the Viscaceae These species have been shown via global analysis of representatives of the entire order to be outside the Nickrent and Franchina (1990) published the first Viscaceae. phylogenetic analysis of parasitic angiosperms using small-subunit 18S rRNA sequences. They showed that For the 18S rDNA data, parsimony analysis using Phoradendron serotinum and Dendrophthora domin­ the branch and bound method yielded a tree of length gensis were apparently derived from the Santalaceae 627. Five clades are present (fig. 15.7A, parts A to E). (represented by a sequence of Buckleya). Since that Clade A consists of the 3 Arceuthobium species; clade time, a large number of complete 18S rDNA sequences B, the 2 Notothixos species; clade C, Korthalsella and have been determined for representatives of all fami­ Ginalloa; clade D, Dendrophthora and Phora­ lies of the Santalales (Nickrent 1992 and unpublished dendron; and clade E, the 2 Viscum species. data). Over 1,000 angiosperm rbcL (ribulose bisphos­ The relationship among Arceuthobium, phate carboxylase/oxygenase, large subunit) Notothixos, and Viscum is unresolved. But with both sequences exist, and their analysis has allowed rbcL and 18S rDNA sequences and high bootstrap val­ unprecedented insight into the phylogeny of higher ues, Korthalsella forms a clade with Ginalloa and plants (Chase and others 1993). Dendrophthora forms a clade with Phoradendron. Sequences of nuclear 18S rDNA and chloroplast Although components of these clades are stable, rbcL genes representing all Viscaceae genera were their relative topology is not. For example, all possible determined to test the effect of phylogeny estimation rearrangements of these clades add only one step to given genes derived from different subcellular com­ the tree, hence the topology is essentially a polytomy. partments and to allow integration into the immense This result is confirmed using bootstrap analysis. rbcL database. Sequences used in this analysis were determined by direct sequencing of products generat­ The rbcL gene is consistently 1,428 bp in length ed via PCR, as discussed above for ITS sequencing. All (476 amino acids), hence manual alignment of the rbcL sequencing primers were supplied by G. sequences is straightforward. The mean percentage of Zurawski (DNAX Research Institute, Palo Alto, CA). sites that differ between Santalum (the outgroup) and The sequence of Phoradendron serotinum was deter- the 7 species ofViscaceae is 5.85% (from 5.39% for

Molecular Systematics 167 Chapter 15

100 Arceuthobium pendens A 98 ...... - Arceuthobium verticil/iflorum Arc. oxycedri * 100 Notothixos leiophyl/us * Notothixos subaureus Korthalsel/a latissima 100 Korthalsel/a lindsayii * Ginalloa arnottiana Dendrophothora domingensis Phoradendron serotinum 95 o Phoradendron californicum Dendrophthora clavata 100 Viscum album Viscum articulatum Antidaphne viscoidea Santalum album

~.Iscum aIbum B 65 Notothixossubaureus Arceuthobium verticil/iflorum 100 100 I Korthalsel/a lin dsayii I 69 Ginal/oa arno ttiana ,..... - 100 I Dendrophotho~a clavata I Phoradendron s erotinum Antidaphne visco ide a Santalum album

Figure 15. 7-Phylograms depicting relationships among genera ofViscaceae, rooted with Antidaphne (Eremolepidaceae) andSantalum (Santalaceae). A: single most parsimonious phylogram (627 steps) derived from anaylses of complete nuclear 18S rDNA. Nodes at asterisks are essentially unresolved, i.e. clades A-E can be rearranged at a cost of 1 step. B: strict consensus of 2 trees (305 steps) derived from analysis of chloroplast rbcL. Bootstrap values listed above the nodes.

168 Molecular Systematics Chapter 15

Korthalsella to 7.3% for Arceuthobium). The accelerat­ its unusually large and non-explosive fruits. The 18S ed evolutionary rate of Arceuthobium is seen by its rDNA and ITS sequence data clearly indicate that this having over twice as many substitutions as Anti­ species is properly placed in Arceuthobium. The loss daphne when compared to the outgroup. Figure of explosive seed dehiscence therefore represents an 15.7B is the strict consensus of 2 trees of length 305 bp evolutionary reversal. with bootstrap confidences indicated at the nodes. Although fewer species were sequenced for the rbcL gene than with 18S rDNA, several relatiopships are Summary of Molecular Evidence concordant. The relationship among Arceuthobium, Notothixos, and Viscum is unresolved; but with both on Phylogenetic Relationships rbcL and 18S rDNA sequences, Korthalsella forms a Morphology, life cycles, and molecular data must clade with Ginalloa and Dendrophthora a clade with be integrated to best estimate the phylogeny of Phoradendron, both with high bootstrap values. Less Arceuthobium species. The following conclusions can support exists for the positions of these major group­ be synthesized from existing information. ings as indicated by the bootstrap value of 65 and 69%. Greater resolution could possibly occur with contin­ The genus ofViscaceae most closely related to ued sampling of Arceuthobium, Notothixos, and Arceuthobium remains unresolved despite analyses of Viscum, but rbcL may not provide sufficient characters 18S rDNA and rbcL sequences. Previous phyletic to distinguish genera within the Viscaceae. Alternative hypotheses that derived Arceuthobium from faster-rate molecules should be examined to resolve Korthalsella are not supported by molecular evidence. relationships among these genera. Division of the genus into two subgenera (Arceu­ thobium and Vaginata) is supported by all analyses. Taken together, results of the 18S rDNA and rbcL analyses support the concept that Phoradendron and From ITS sequence data, the New World members Dendrophthora are closely related and that Viscum is of subgenus Arceuthobium comprise two groups likely the most primitive member of the genus (Wiens (fig. 15.6). The New World Arceuthobium abietis­ and Barlow 1971). It does not appear, however, that religiosae is genetically very similar to the Old World Korthalsella is the sister taxon to Arceuthobium nor A. oxycedri and included with A. juniperiprocerae in that Ginalloa and Notothixos are closely related. The section Arceuthobium (table 15.2). Arceuthobium topology of the phylogenetic tree suggested by Wiens abietis-religiosae is genetically distinct from other New and Barlow (1971) is as follows: (((Ginalloa, World members of the subgenus Arceuthobium Notothixos) Viscum) ((Korthalsella, Arceuthobium) (A. americanum and A. verticillijlorum) that comprise (Phoradendron, Dendrophthora))). This arrangement the newly proposed section Americana (table 15.2). of genera, however, results in an 18S rDNA tree 29 This high amount of genetic differentiation is also seen steps longer than the minimum length, hence it is between A. oxycedri and both A. verticillijlorum and unlikely that this topology best represents the phy­ A. pende.ns when the more conservative 18S rDNA logeny. Chromosome size and number may not be as sequences are compared. Further sampling of ITS useful for inferring phylogenetic relationships in variation in other Old World members of subgenus Viscaceae as in Loranthaceae. Arceuthobium is needed. The evidence for linking Korthalsella with The reconstituted subgenus Vaginata, section Arceuthobium has been the reported presence of Vaginata (table 15.2), includes taxa previously in sec­ "mildly explosive" fruits in Korthalsella. However, tion Vaginata (Arceuthobium aureum, A. durangense, these fruits may either not really be explosive A. gil/ii, A. globosum, A. hawksworthii, A. nigrum, A. (Molvray, personal communication; Nickrent, unpub­ vaginata, and A. yecorense) as well as several species lished data) or may represent another case of conver­ previously placed in section Campylopoda (A. divari­ gence. The molecular analyses did not indicate which catum, A. oaxacanum, A. rubrum, and A. strictum) genus is the closest relative of Arceuthobium. and in section Minuta (A. douglasil). Biogeographic evidence suggests Notothixos or Neither isozyme nor DNA analyses cluster Viscum, because both have centers of diversity (and Arceuthobium douglasii with A. pusillum, but the presumably centers of origin) in Asia, as was also pos­ association of A. douglasii with A. divaricatum tulated for Arceuthobium (Hawksworth and Wiens receives strong support from isozyme data and moder­ 1972). ate support from ITS-l sequence data. Section Minuta Wiens (personal communication) questioned should be reconstituted as series Minuta composed of whether Arceuthobium verticillijlorum should be seg­ A. douglasii and A. divaricatum (table 15.2). regated as a separate genus from Arceuthobium given

Molecular Systematics 169 Chapter 15

TABLE 15.2 - Classification of Arceuthobium M. Bieb. based on molecular systematics

Subgenus Arceuthobium Section Arceuthobium Caules cum frequentibus vel infrequentibus verticillatis ramis secundariis; surculi principales vulgo 3-16 cm alti et basi 1-10 mm diametro; flores staminati plerumque 3 meri; anthesis vernalis vel autumnalis. Type species: A. oxycedri (DC.) M. Bieb. 1. A. abietis-religiosae Heil 2. A.juniperi-procerae Chiovenda* 3. A. oxycedri (DC.) M. Bieb.

Section Americana Nickrent [Sec. Nov.] Caules cum vel sine verticillatis ramis secundariis; surculi principales vulgo 6-7 cm alti et basi 1-5 mm diametro; flores staminati 3 vel 4 meri; anthesis vernalis; fructus auturnno maturant plus quam 15 menses post pollinationem; parasiti praecipue in Pinus. Type species: A. ameri­ canum Nutt. ex Engelm. 4. A. americanum Nutt. ex Enge1m. 5. A. verticilliflorum Engelm.

Section Azorica Nickrent [Sec. Nov.] Caules cum infrequentibus verticillatis ramis secundariis; surculi principalis 7-14 cm alti et basi 5-9 mm diametro; flores staminati plerumque 4 meri; flores pistillatae verticillatis dispositae et cum bractea subtentae; inJuniperus brevi/olia parasiticum. Type species: A. azoricum Hawksw. & Wiens 6. A. azoricum Hawksw. & Wiens*

Section Chinense Nickrent [Sec. Nov.] Caules cum vel sine verticillatis ramis secundariis; surculi principales 0.5-22 cm aiti et basi 1-3 mm diametro; flores staminati 3 vel 4 meri; anthesis vernalis vel autumnalis; efficiens infectus systemicos in Keteleeria, Pinus, Picea et Abies. Type species: A. chinense Lecomte 7. A. chinense Lecomte* 8. A. minutissimum J.D. Hooker 9. A. pini Hawksw. & Wiens* 10. A. sichuanense (H. S. Kiu) Hawksw. & Wiens* 11. A. tibetense H. S. Kiu & W. Ren*

Subgenus Vaginata Hawksw. & Wiens Section Penda Nickrent [Sec. Nov.] Cauies flabellatim ramosi secundarii; surculi principales 1-22 cm alti et basi 1.5-3.5 mm diametro; flores staminati 2 vel 3 meri; anthesis mense Septembri; fructus Septembri maturant 12 menses post pollinationem; efficiens infectus systemicos in Pinus. Type species: A. pendens Hawksw. & Wiens 12. A. guatemalense Hawksw. & Wiens 13. A. pendens Hawksw. & Wiens

Section Vaginata Series Globosa Nickrent [Ser. Nov.] Cauies flabella tim ramosi secundarii; surculi principaies 10-22 cm aiti et basi 3-48 mm diametro; flores staminati 3 vei4 meri; tempora anthesis et fructificandi variabilia vel continua; parasiti praecipue in Pinus. Type species: A. globosum Hawksw. & Wiens 14. A. aureum Hawksw. & Wiens subsp. aureumt 15. A. aureum Hawksw. & Wiens subsp.petersonii Hawksw. & Wiens 16. A. globosum Hawksw. & Wiens subsp. globosum 17. A. globosum Hawksw. & Wiens subsp. grandicaule Hawksw. & Wiens

170 Molecular Systematics Chapter 15

TABLE 15.2 - Classification ofArceuthobium M. Bieb. based on molecular systematics (continued)

Subgenus Vaginata Hawksw. & Wiens (continued) Section Vaginata (continued) Series Rubra Hawksw. & Wiens 18. A. gillii Hawksw. & Wiens 19. A. nigrum (Hawksw. & Wiens) Hawksw. & Wiens 20. A. oaxacanum Hawksw. & Wiens· 21. A. rubrum Hawksw. & Wiens

Series Vaginata Syn. Series Stricta Hawksw. & Wiens (Brittonia 22:266, 1970) 22. A. durangense (Hawksw. & Wiens) Hawksw. & Wiens 23. A. hawksworthii Wiens & c. G. Shaw lIlt 24. A. strictum Hawksw. & Wiens 25. A. vaginatum (Willd.) Presl subsp. vaginatum 26. A. vaginatum (Willd.) Presl subsp. cryptopodum (Engelm.) Hawksw. & Wiens 27. A. yecorense Hawksw. & Wiens

Series Minuta Hawksw. & Wiens 28. A. divaricatum Engelm. 29. A. douglasii Engelm.

Section Pusilla Nickrent [Sec. Nov.] Caules flabellatim ramosi secundarii si adsunt; surculi principales 1-21 cm alti et basi 1-9 mm diametro; flores staminati vulgo 3 meri, aliquan­ do 2 vel 4 meri; lobi perianthii cum rubra interna superficie; anthesis vernalis vel autumnalis. Type species: A. pusillum Peck 30. A. bicarinatum Urban 31. A. hondurense Hawksw. & Wiens· 32. A. pusillum Peck

Section Campylopoda Hawksw. & Wiens 33a. A. abietinum Engelm. ex Munz f. sp. concoloris 33b. A. abietinum Engelm. ex Munz f. sp. magnificae 34. A. apachecum Hawksw. & Wiens 35.· A. blumeri A. Nelson 36. A. californicum Hawksw. & Wiens 37. A. campylopodum Engelm. 38. A. cyanocarpum (A. Nelson ex Rydberg) Coulter & Nelson 39. A. laricis (Piper) St.John 40. A. littorum Hawksw" Wiens & Nickrent 41. A. microcarpum (Engelm.) Hawksw. & Wiens 42. A. monticola Hawksw., Wiens & Nickrent 43. A. occidentale Engelm. 44. A. siskiyouense Hawksw., Wiens & Nickrent 45. A. tsugense (Rosendahl) G. N.Jones subsp. tsugense 46. A. tsugense (Rosendahl) G. N. Jones subsp. mertensianae Hawksw. & Nickrent

*Taxa not examined using molecular methods, hence placement is tentative. tRecently obtained ITS and 5.8S rDNA sequences indicate that A. aureum subsp. aureum is closely related to A. globosum and that A. hawksworthii is related to A. vaginatum.

Molecular Systematics 171 Chapter 15

Arceuthobium pusillum is genetically very similar logical but low DNA divergence. Clarkia ligulata and to A. bicarinatum but has developed a large number C. biloba are given as examples for the first syndrome. of morphological and physiological apomorphies In Arceuthobium, a similar situation occurs among such as reduced shoot size and systemic broom forma­ members of section Campylopoda. The second syn­ tion. These two species and A. hondurense are the drome was illustrated by comparing sections likely survivors of an ancient lineage whose most Myxocarpa and Eucharidum of Clarkia or the Old recent common ancestor became extinct during the World section Skinnera with New World sections. In Tertiary Period. These species are recognized as Arceuthobium, the most morphological divergence is members of section Pusilla (table 15.2). seen between representatives of subgenera Arceuthobium and Vaginata. This distinction is Two additional segregates, Arceuthobium pen­ strongly supported by molecular evidence. The third dens and A. guatemalense, from the former section syndrome can be demonstrated by comparing C. ros­ Campylopoda are deserving of placement in the new trata with C. lewisii and C. cylindrica. In dwarf mistle­ section Penda. These species are narrow endemics toes, a low amount of morphological divergence is and are also likely relicts of a common Tertiary seen between A. pendens and A. guatemalense as ancestor. compared with species in section Campylopoda The remaining section Campylopoda now com­ (however, DNA evidence indicates these two groups prises 13 species mainly of the United States and with a are not closely related). Finally, the fourth syndrome high degree of morphological and genetic similarity. can be seen in comparisons of Clarkia rostrata and c. The reconstituted section includes Arceuthobium abi­ epilobioides which differ in breeding system and floral etinum, A. apachecum, A. blumeri, A. calijornicum, morphology but are extremely similar genetically. A. campylopodum, A. cyanocarpum, A. laricis, A. litto­ Similarly, Heterogaura heterandra and C. dudleyana rum, A. microcarpum, A. monticola, A. occidentale, exhibit this type of discordance. For Arceuthobium, A. siskiyouense, and A. tsugense. this syndrome is best shown by A. pusillum and A. bicarinatum or by A. douglasii and A. divaricatum. The observations made by Sytsma and Smith (1992) regarding concordance or discordance The dwarf mistletoes continue to present a chal­ between morphological" and molecular divergence in lenge to the systematist, as do all parasitic plants that Clarkia and Fuchsia are applicable to dwarf mistle­ follow reductional and/or convergent evolutionary toes. Basically, four syndromes were described: low paths. These plants provide the ultimate test of our morphological and molecular divergence, high mor­ abilities to reconstruct phylogenies, therefore alternate phological and high DNA divergence, low morpho­ data sets for other genes must be assembled to confirm logical but high DNA divergence, and high morpho- or support proposed relationships.

172 Molecular Systematics