Chapter 15—Molecular Systematics

Daniel L. Nickrent*

*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).

 

Despite thousands of references to dwarf mistletoes, relatively little work has been conducted on the systematics or phylogenetics of the genus. Molecular analyses of intergeneric or interspecific relationships are even fewer. This chapter reviews previous studies that used macromolecular characters in systematic studies of Arceuthobium, presents new data, and summarizes those relationships that appear to be most strongly supported.

Macromolecular characters that have been examined within the dwarf mistletoes include isozymes and DNA sequences from nuclear ribosomal DNA and a plastid-encoded gene (rbcL). Using isozyme characters of triploid seed endosperms, interspecific relationships have been determined for 25 New World species, and the results are reviewed below. Subsequent work (Nickrent and Butler 1990, Nickrent and Stell 1990, Nickrent and Butler 1991) on species complexes within section Campylopoda utilized diploid shoot tissue, thereby allowing for the use of a wider array of population genetics statistics. The genetically variable internal transcribed spacer (ITS) regions have been sequenced for 22 species of New World and 1 species of Old World dwarf mistletoe (Schuette 1992, Schuette and Nickrent 1992, Nickrent and others 1994). Results of analysis of these sequences are compared with those derived from isozymes and other characters. Genetically conservative small subunit (18S) ribosomal DNA sequences for 3 species of Arceuthobium and representatives of the other 6 genera of Viscaceae have been determined. Intergeneric relationships are addressed using these 18S rDNA sequences and are compared to a similar study employing the chloroplast gene rbcL.

 

Isozyme Analyses of Interspecific Relationships

Isozyme electrophoresis has been extensively used to address questions of population genetics, breeding systems, and systematic relationships in plants (Soltis and Soltis 1989). Because of the extreme reduction associated with the parasitic habit and the resulting paucity of morphological characters, isozyme electrophoresis has proven especially valuable in providing useful data for examining species relationships in Arceuthobium. Nickrent and others (1984) and Nickrent (1986) first examined the isozymes of 19 North American taxa of Arceuthobium. From that study, it was learned that the genus has remarkably high levels of genetic diversity—67% of the loci were polymorphic and averaged 2.23 alleles per locus. This result was surprising given the relative homogeneity of the genus with regards to morphology and flavonoid composition (chapter 14).

Many of the results of that isozyme study were consistent with the taxonomic classifications of Hawksworth and Wiens (1972, 1984, and table 14.1), including the recognition of 2 subgenera (Arceuthobium and Vaginata), the close relationship among species of section Campylopoda, and the clustering of A. gillii (sensu lato) and A. vaginatum (sensu lato). Isozyme analysis did not, however, support placement of A. douglasii and A. pusillum together in section Minuta. An unexpected result of that study was the grouping of A. douglasii, a parasite of Pseudotsuga menziesii, with A. divaricatum, a parasite of pinyons. That isozyme analysis clearly raised as many questions as it resolved, hence further work was needed, especially on Mexican and Central American taxa.

The following portion of this chapter presents methodologies and results of an electrophoretic study of 36 dwarf mistletoe populations. The analysis incorporates genetic data on 13 populations of 7 Mexican taxa that were not included in the previous isozyme study (Nickrent 1986) but are pivotal for understanding species and sectional relationships. Some questions remained after the 1986 study:

Methodologies

Electrophoretic methods are essentially as reported in Nickrent (1986). Briefly, seeds are germinated in H2O2; viscin is removed from the endosperm; and the endosperm is stored at -70°C. Endosperms are then homogenized in an extraction buffer; the buffer absorbed onto filter paper wicks; and the wicks inserted into horizontal starch gels. Enzyme separation is effected by electrophoresis, and the gel is sliced and stained for different enzymes (Wendel and Weeden 1989). The resulting banding patterns are used to infer genotypes. Results of gel electrophoresis for 14 individuals of 2 species of Arceuthobium stained for 6-phosphogluconate dehydrogenase appear in figure 15.1A. Relative intensities of the bands in one lane for heterozygotes (4:4:1 or 1:4:4) confirm the triploid nature of the endosperm tissue. In this study, 7 enzyme systems were used, representing 9 putative enzyme loci: 6-PGD, IDH, GPI, ADH-1, ADH-2, GDH, G-6-PDH, MDH-2, and MDH-3.

Genetic interpretation follows that reported in Nickrent (1986). Because the genotypes were not diploid, individual genotypes could not be entered directly into the statistical program BIOSYS-1 (Swofford and Selander 1981). Instead, allele frequencies were determined for all populations and loci, and these frequencies used as input data.

Isozyme data for 19 taxa (Nickrent 1986) were used in combination with new data for 13 populations of 7 additional taxa (Arceuthobium abietis-religiosae, A. globosum subsp. grandicaule, A. nigrum, A. pendens, A. strictum, A. vaginatum subsp. vaginatum, and A. verticilliflorum), yielding a total of 26 taxa (see the appendix "Specimens Examined" for additional data on host and collection locality).

 

Results and Discussion

Genetic variability statistics for the 36 dwarf mistletoe populations appear in table 15.1. The overall mean percentage of polymorphic loci is 48.7%, and the mean number of alleles per locus is 2.15. These values are lower than those reported in Nickrent (1986) because several populations of Mexican dwarf mistletoes with unusually low levels of polymorphism were included—Arceuthobium abietis-religiosae, A. globosum subsp. grandicaule, A. pendens, A. rubrum, A. strictum, and A. verticilliflorum. The mean number of alleles per locus for these 6 taxa (1.5) is lower than the overall mean for the genus (2.15); this reduction in allele frequency may be a consequence of their restricted distributions. However, two other Mexican taxa, A. vaginatum subsp. vaginatum and A. durangense, show high levels of polymorphism that are comparable to levels found in a related and more northern taxon, A. vaginatum subsp. cryptopodum. This contrast in species-to-species variation in polymorphism agrees with conclusions of Nickrent (1986) and Hawksworth and Wiens (1972) that section Vaginata (which includes A. vaginatum and A. durangense) is genetically and morphologically more variable than other sections in the genus. Although A. globosum subsp. grandicaule shows a low level of polymorphism and is also in section Vaginata, only 30 individuals from one population were examined electrophoretically, hence this sample may not be representative of the species’ genetic diversity.

Interpopulational genetic differences were calculated using the chord distance of Cavalli-Sforza and Edwards (1967). These distances were then used for UPGMA (Sneath and Sokal 1973) phenogram construction (fig. 15.2). Values for several combinations are very nearly or equal to 1.0, indicating that few or no alleles are shared between the populations being compared. These large distance values generally resulted from comparisons among members of subgenus Vaginata section Campylopoda and subgenus Arceuthobium. This result is not unexpected because subgenus Arceuthobium contains 3 taxa (A. abietis-religiosae, A. americanum, and A. verticilliflorum) that are morphologically and genetically distinct from the rest of the genus.

 

Section Campylopoda

Eleven of the taxa examined are included within section Campylopoda, series Campylopoda; these taxa grouped together at a chord distance of 0.45 or less (fig. 15.2). Two additional taxa of section Campylopoda (as delimited in table 14.1) are newly analyzed—Arceuthobium pendens and A. strictum. Although they cluster together, the populations are linked at a genetic distance value of 0.54, hence their relationship is distant. The branch comprising these 2 taxa joins the 11 other taxa of section Campylopoda at the 0.65 level, indicating a significant amount of genetic differentiation. Arceuthobium strictum and A. pendens are 2 of the most unusual dwarf mistletoes found in Mexico and both have limited distributions: A. strictum is a parasite of Pinus leiophylla and P. teocote and is found only in the Sierra Madre Occidentale and in the state of Durango. Arceuthobium pendens is known only from Puebla, San Luis Potosí, and Veracruz and like A. divaricatum parasitizes pinyons. Although Hawksworth and Wiens (1980) placed both A. pendens and A. divaricatum in section Campylopoda series Campylopoda, several morphological features and flavonoid chemistry (table 14.2) gave preliminary evidence that A. pendens was quite distinct from A. divaricatum. This isozyme evidence is in agreement that these two parasites of pinyons are not closely related.

Placement of Arceuthobium pendens and A. strictum in a position intermediate between sections Campylopoda and Vaginata (fig. 15.2) indicates that a gradation of evolutionary divergence may exist. The extremes of this gradient are represented by the "strict" Campylopoda taxa of the western United States and the "strict" Vaginata taxa of Mexico. Interestingly, the most distant members of series Campylopoda are A. apachecum and A. blumeri; these species occur in the Southwest and northern Mexico. Whether this observation reflects a "genotypic cline" resulting from introgression or divergence from a common genotype is not known.

 

Arceuthobium divaricatum and A. douglasii

Arceuthobium douglasii is most closely allied (fig. 15.2 and Nickrent 1986) with A. divaricatum, not with A. pusillum (section Minuta of Hawksworth and Wiens 1972). This relationship is unexpected because the principle hosts of A. douglasii (Pseudotsuga menziesii) and A. divaricatum (pinyons) are not closely related. These mistletoes are morphologically distinct, but A. douglasii is so morphologically reduced that association with any taxon is obscure. The concept of biochemical convergence between these species was discussed by Nickrent (1986) and dismissed in favor of a more parsimonious explanation of genetic and phylogenetic similarity. These species share 16 alleles across 6 loci, and both species are fixed for GDH66 (an isozyme absent in all other species). Both these mistletoes were included in section Vaginata by Nickrent (1986); however, addition of other Mexican taxa in the present analysis suggests that these species more likely form a transitional group between sections Vaginata and Campylopoda.

Evidence from flowering groups (Wiens 1968 and chapter 14) also supports the proposed relationship between Arceuthobium douglasii and members of section Vaginata.

Although some species of section Vaginata (e.g., A. durangense, A. globosum, and A. vaginatum) have direct spring-flowering, other species of section Vaginata (e.g., A. gillii and A. nigrum) and A. douglasii have indirect spring-flowering. Nickrent (1984) presents evidence supporting a derivation of indirect spring-flowering from direct spring-flowering as a response to exposure to cooler climates.

During these isozyme analyses, thousands of dwarf mistletoe seeds have been examined. Several seed features appear to be useful in differentiating between sections Campylopoda and Vaginata. Seeds of Vaginata species are often ellipsoid (versa pyriform to ovoid), have a slight protuberance at the chalazal end, have short viscin strands arranged more densely near the radicular end, and have endosperm epidermal cells pigmented with red (or purple/maroon) or variegated green and red (pinto). Because Arceuthobium douglasii seeds are ellipsoid and display the pinto phenotype (A. pusillum seeds are pyriform and entirely green), the species’ affinity to section Vaginata is difficult to ignore.

 

Section Vaginata

The new taxa and populations of the predominantly Mexican section Vaginata included in this study are Arceuthobium durangense, A. globosum subsp. grandicaule, A. nigrum, and A. vaginatum subsp. vaginatum. Similarity averaging shows that section Vaginata, as defined here, is second only to subgenus Arceuthobium in terms of within-group heterogeneity (fig 15.2).

Arceuthobium globosum subsp. grandicaule emerges as the most distinct member of section Vaginata, as it had from previous morphological and physiological studies (Hawksworth and Wiens 1972, 1984). This taxon has the largest shoot diameter of the genus (reaching nearly 5 cm), parasitizes at least 12 host species, and ranges as far south as Guatemala. Samples from populations of A. globosum subsp. globosum (a more northern subspecies) are needed to determine levels of genetic similarity between the 2 taxa currently treated as suspecies.

The phylogenetic position of Arceuthobium rubrum remains somewhat enigmatic because this taxon clusters (distantly) with members of section Campylopoda based on phenetic analysis of morphological data (Hawksworth and Wiens 1972) but the taxon is clearly a component of the Vaginata group based on isozymes. Arceuthobium rubrum clusters at a genetic distance of 0.6 (fig. 15.2) with A. gillii and A. nigrum of section Vaginata. In a cladistic analysis of morphological and physiological features (Nickrent 1984), A. rubrum was identified as the most basal taxon of the Campylopoda clade; its morphological similarities to section Campylopoda may only be the result of convergence. Seeds of A. rubrum are ellipsoid and pigmented either green, maroon, or pinto, therefore geographic distribution, isozyme evidence, and seed characters support placement of A. rubrum in section Vaginata.

Arceuthobium gillii and A. nigrum were originally classified as sister subspecies (Hawksworth and Wiens 1972). In this study, they clustered together at a genetic distance of 0.553 and stand apart from the rest of section Vaginata (fig. 15.2). Although only two populations were analyzed, these taxa appear sufficiently differentiated to justify their elevation to specific status.

Clustering of taxa related to Arceuthobium vaginatum (A. vaginatum subsp. vaginatum, A. vaginatum subsp. cryptopodum, and A. durangense) corresponds to the existing taxonomy for some populations (e.g., populations 30, 31, 32, and 34) but appears genetically distinct for other populations (e.g., populations 15, 16, and 17). The lack of clustering for populations of A. durangense is partly due to the high number of alleles present at the GPI locus. Population 17 (Puerto Buenos Aires) has 8 alleles at this locus, whereas population 16 (El Madroño, only 7 km away) shows just 3 alleles at this locus. Although sample sizes for these populations differ, the results provide evidence for significant genetic differentiation over relatively small areas.

 

Arceuthobium pusillum

As reported in Nickrent (1986), isozyme evidence indicates that Arceuthobium pusillum is a distinct taxon in the genus that is placed midway between subgenus Vaginata and Arceuthobium. Isozyme analysis does not support a section Minuta composed of A. pusillum and A. douglasii. These results agree with those of Crawford and Hawksworth (1979 and see table 14.2), which show that these two species have different flavonoid patterns. Isozyme data also do not provide evidence that A. pusillum and A. douglasii are recent derivatives from section Campylopoda as suggested by Hawksworth and Wiens (1972, page 38). That these species share isophasic broom development and indirect spring-flowering must be interpreted as convergence.

 

Subgenus Arceuthobium

Only Arceuthobium americanum represented subgenus Arceuthobium in the earlier isozyme study (Nickrent 1986). With the inclusion of A. abietis-religiosae and A. verticilliflorum, all three New World members of the subgenus have been examined electrophoretically. These species share several isozymes (IDH136, GPI200, G-6-PGD100, and MDH-3187) and occupy the same branch of the phenogram (fig. 15.2). These results demonstrate that they are at least distantly related. A comparison of genetic distance values indicate that A. americanum is more closely related to A. verticilliflorum than it is to A. abietis-religiosae. The two populations of A. abietis-religiosae showed a genetic distance of 0.452; this relatively high value indicates that substantial genetic differentiation has occurred between populations separated by only 400 km. This genetic differentiation is further demonstrated by the fixation for different alleles at the IDH locus (IDH136 and IDH160). According to similarity/distance measure averaging, subgenus Arceuthobium is the most heterogeneous of all the dwarf mistletoe groups examined, and A. abietis-religiosae occupies the most basal position among the species examined.

 

Conclusions

With the new isozyme evidence, questions raised in the introduction can now be answered. Arceuthobium strictum is distantly related to most other taxa in section Campylopoda and clusters with A. rubrum. These relationships are not totally in conflict with the classifications of Hawksworth and Wiens (1972 and table 14.1), in which these species were placed in their own series (Stricta and Rubra, respectively). It appears, however, that the majority of section Campylopoda species are found in the United States, where as section Vaginata species predominate in Mexico.

The two parasites of pinyons, Arceuthobium divaricatum and A. pendens, are not closely related. This conclusion is supported by the isozyme data, markedly different flavonoid chemistry, systemic broom formation in A. pendens, and different hosts (Hawksworth and Wiens 1980).

Arceuthobium gillii and A. nigrum are clearly related but cluster at a genetic distance value of 0.553, thus supporting their recognition as distinct species. The relatives of A. vaginatum have high levels of genetic diversity, and cluster analysis indicates a substantial genetic differentiation between populations. Further work is required to better understand apportionment of genetic variation in the Arceuthobium vaginata complex.

Subgenus Arceuthobium shows greater within-group heterogeneity than any of the other groups. Despite the variation, isozyme analysis places Arceuthobium abietis-religiosae, A. americanum, and A. verticilliflorum on a single branch that joins the remainder of the species at a genetic distance of 0.82. Thus, isozyme data support use of verticillate secondary branching (Mark and Hawksworth 1981) as a distinguishing character for the subgenus.

 

Species Relationships in the Arceuthobium campylopodum Complex

Additional isozyme analyses have been conducted on various members of the Arceuthobium campylopodum complex (Nickrent and Butler 1990, Nickrent and Stell 1990, Nickrent and Butler 1991). These studies utilized diploid shoot tissue as a source of isozymes, hence a larger number of population genetic analyses could be conducted. Enzyme banding patterns typical of diploid tissue are shown in fig. 15.1B. Based upon UPGMA cluster analysis, California coastal populations of Arceuthobium parasitic on Pinus radiata and P. muricata are genetically distinct from A. campylopodum (sensu stricto) and are segregated at the specific level as A. littorum. Examination of isozymes from allopatric and sympatric populations of A. campylopodum and A. occidentale did not reveal significant genetic differentiation, hence they could be considered a single biological species.

Nickrent and Butler (1991) examined genetic relationships among dwarf mistletoes related to Arceuthobium campylopodum that are parasitic on Pinus attenuata and P. monticola of northwestern California and southwestern Oregon. The parasites of P. attenuata and P. monticola were genetically distinct from each other. These species were named, respectively, A. siskiyouense and A. monticola (Hawksworth and others 1992b). This result is in accordance with the observation that the Klamath–Siskiyou Mountain region contains a highly diverse and endemic flora. These dwarf mistletoes likely represent the most recent evolutionary lines to diverge from the A. campylopodum complex.

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 mertensiana is genetically distinct and deserving of taxonomic recognition as A. tsugense subsp. mertensianae (Nickrent and Stell 1990, Hawksworth and others 1992b). Arceuthobium tsugense subsp. tsugense consists of two morphologically similar host races parasitic on Tsuga heterophylla (western hemlock race) and Pinus contorta subsp. contorta (shore pine race). Isozyme analysis failed to detect significant genetic differentiation between these two host–forms, hence they were retained within the same subspecies.

 

Species Relationships From Ribosomal DNA Spacer Sequences

Many studies addressing interspecific relationships in plants have been conducted using restriction site data from chloroplast DNA (see Soltis and others 1992). Few studies have used DNA sequences to examine interspecific relationships because a gene or segment of DNA that is of adequate size and that evolves at a sufficiently fast rate is required. Recently, the internal transcribed spacer (ITS) regions of the ribosomal DNA cistron have been shown to evolve at rates appropriate for examining more recently diverging lineages (Baldwin 1993). As shown in fig. 15.3, the rDNA cistron (transcriptional unit) comprises (from 5´ to 3´) an external transcribed spacer (ETS), the 18S rDNA, ITS-1, 5.8S rDNA, ITS-2, 26S rDNA, and a transcription termination site (TTS). This cistron is tandemly repeated several hundred to several thousand times in the nucleolar organizing region (Arnheim 1983).

The transcriptional units themselves are separated by a nontranscribed spacer (NTS). The NTS region has been used to study variation at the population or even individual level (May and Appels 1987). Although ribosomal cistrons occur in high copy number, the sequence of each repeat within a genome is conserved via unequal crossing over and other DNA turnover mechanisms. This phenomenon is referred to as "horizontal" or "concerted evolution" (Brown and others 1972, Arnheim and others 1980, Arnheim 1983). Within the cistron, ribosomal genes and spacers show markedly different tempos of evolution (Jorgansen and Cluster 1988), hence studies of closely related and distantly related organisms can be conducted using the appropriate region. The ITS regions and enclosed 5.8S rDNA were sequenced and analyzed in 22 species of Arceuthobium to allow a comparison with phylogenies generated from other methods.

 

Methodologies

Sequence analyses were conducted on samples from 24 populations of Arceuthobium that represented 22 taxa (see fifth appendix, "Specimens Examined"). Both shoot and seed materials were used as sources for DNA. Genomic DNA was obtained from shoots by grinding in liquid nitrogen and extracting in 2x CTAB (Doyle and Doyle 1987). Crude homogenates of seeds were made in a buffered protease solution (Schuette 1992, Nickrent and others 1994).

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 sites on both the 18S and 26S rDNA allow the construction of forward and reverse primers that bracket ITS-1, 5.8S rDNA, and ITS-2. A common primer combination employed the 18S 1830 forward and 26S 25 reverse (5´ AACAAGGTTTCCGTAGGTGA-3´ and 5´ TATGCTTAAAYTCAGCGGGT-3´ respectively), which yielded a 0.64-kb fragment upon symmetrical amplification. This fragment was used as a template for an asymmetrical PCR reaction (Gyllensten and Erlich 1988) that produces single-stranded DNA. In some cases, the double-stranded template was sequenced directly. Because Arceuthobium abietis-religiosae and A. oxycedri apparently had mutations at one or both of the above two priming sites, alternate internal 18S (forward) and 26S (reverse) primers were used. The PCR product was then gel purified for subsequent sequencing reactions (Nickrent 1994). These sequencing reactions were carried out using the terminal amplification primers or forward and reverse primers constructed for the conserved sites on the 5.8S rDNA (sites 32 to 52). Chain-termination sequencing reactions using dideoxynucleotides (Sanger and others 1977) were conducted using Sequenase® (U.S. Biochemical Corp.).

An autoradiogram with ITS sequences for three dwarf mistletoe species is shown in figure 15.4. Most mutations were base substitutions, thus allowing manual alignment using "Eyeball Sequence Editor" (Cabot and Beckenbach 1989). Minimum length trees and other analyses of the aligned sequences were conducted using PAUP version 3.1 (Swofford 1993).

 

Results and Discussion

Overall, little length variation occurs. ITS-1 sequences varied by less than 1% from the average length (208 base pairs); ITS-2 varied by less than 1.5% when compared to the average (226 bp). The 5.8S rDNA sequences were usually 167 bp in length, although the three members of subgenus Arceuthobium and Arceuthobium divaricatum had a 5.8S rDNA of length 166 bp. This is slightly larger than the common length (164 bp) seen in other angiosperms. The ITS sites can be classified according to their variability: 39% are monotypic, 33% are ditypic, 15% are tritypic, 2% are tetratypic, and 11% are insertions or deletions. These values were determined without the inclusion of A. abietis-religiosae and A. oxycedri because of their high sequence divergence compared with other taxa (see below). In Arceuthobium, ITS-1 is less variable than ITS-2 and is also smaller (which is the case for many other, mainly asterid, species). At 208 bp, dwarf mistletoe ITS-1 is within the range (194 to 265 bp) reported for other angiosperms (Baldwin 1993). In Calycadenia and other composites, as well as Vicia and Sinapis, ITS-1 is larger than ITS-2 (Baldwin 1993).

The alignment of ITS-1, 5.8S rDNA, and ITS-2 produced a matrix of 22 Arceuthobium species by 619 sites (a portion is shown in fig. 15.5). Of these sites, 388 (62.6%) were variable. Because A. abietis-religiosae and A. oxycedri had high sequence divergence 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 (i.e., a different nucleotide shared by at least 2 taxa). ITS-1 contained 71 sites (40.8% of total); 5.8S had 15 sites (8.6%); and ITS-2 had 88 sites (50.6%). When the number 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-1, 46.8% (15/32) for 5.8S, and 63.7% (88/138) for ITS-2.

To test for possible significant intraspecific variation, partial ITS sequences (100 bp) were determined for individuals from 3 populations of Arceuthobium americanum and 2 populations of A. divaricatum. A single change was detected in A. americanum, and no differences were seen in A. divaricatum. Several taxa have been sequenced twice from either the same genomic DNA sample or a second crude seed extract. Only very rarely were true polymorphisms detected, and these were subsequently coded in the matrix as ambiguous nucleotides.

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 the reliability of the resulting clades (fig. 15.6). As expected from their large sequence divergence, Arceuthobium abietis-religiosae and A. oxycedri form a clade well removed from the remainder of the genus. Removing these species from the analysis and substituting A. americanum and A. verticilliflorum as outgroups resulted in the same topologies. Specific relationships derived from this analysis are discussed below.

Section Campylopoda

As evidenced from the clustering and branch lengths, four members of section Campylopoda are genetically very similar—A. abietinum f. sp. magnificae, A. apachecum, A. campylopodum, and A. microcarpum. Sequences of A. cyanocarpum, A. occidentale, and A. tsugense were either very similar or identical to those of the above four taxa and therefore not included. The section Campylopoda clade, comparable to series Campylopoda (as delimited by Hawksworth and Wiens 1972, 1984, and table 14.1), occurs mainly in the United States and does not include Mexican and Caribbean species such as A. guatemalense, A. pendens, A. rubrum, A. bicarinatum, and A. strictum. The latter three of these species were previously segregated into series Rubra (A. rubrum and A. bicarinatum) and Stricta (A. strictum) (Hawksworth and Wiens 1972 and table 14.1), thus providing some indication of their differentiation from series Campylopoda. ITS sequence analysis does not support a close relationship among members of these three series; but rather, the analysis indicates various relationships to members of section Vaginata.

A strongly supported result of the ITS analysis (100% bootstrap) is the association of Arceuthobium guatemalense with A. pendens. This clade appears basal to all other members of subgenus Vaginata, not as a component of section Campylopoda, series Campylopoda (as delimited by Hawksworth and Wiens 1984 and table 14.1). Arceuthobium guatemalense is confined to the mountains of Guatemala and southern Mexico, where it parasitizes Pinus ayacahuite (of subgenus Haploxylon). Arceuthobium pendens is known only from Puebla, San Luis Potosí, and Veracruz, Mexico, and is parasitic on the Haploxylon pines, P. discolor and P. orizabensis. Both of these mistletoes and their hosts are narrow endemics. Given their position on the phylogram and their endemic distributions, these species could represent relictual taxa that diverged early during the migration and evolution of Arceuthobium in the New World.

 

Arceuthobium divaricatum and A. douglasii

Although classified into different sections by Hawksworth and Wiens (1972 and table 14.1), Arceuthobium divaricatum and A. douglasii cluster together according to isozyme analysis (Nickrent 1986). Arceuthobium divaricatum occurs near the base of section Vaginata and near A. globosum in the derived phylogram (fig. 15.6). When ITS-1 alone is analyzed (tree not shown), A. divaricatum and A. douglasii cluster together with a high bootstrap confidence. Examination of the alignment shows that A. divaricatum has a divergent ITS-2 sequence compared to other members of subgenus Vaginata. Taking the isozyme and ITS evidence together, a clade composed of A. divaricatum and A. douglasii is presently favored.

Section Vaginata

Two strongly supported clades representing section Vaginata are seen following bootstrap analysis (fig. 15.6). The first is composed of Arceuthobium vaginatum subsp. vaginatum, A. vaginatum subsp. cryptopodum, A. durangense, and A. strictum. Previous classifications (Hawksworth and Wiens 1972) and isozyme studies (Nickrent 1986 and this chapter) have shown relationships among the first three of these taxa. The addition of A. strictum is somewhat surprising, although isozyme analysis had shown genetic divergence between it and species in series Campylopoda. All four of these taxa parasitize pines of subgenus Diploxylon and their distributions range from the northern Sierra Madre Occidental (Durango through Chihuahua and Sonora) to the southwestern United States (Arizona, New Mexico).

The second clade is composed of Arceuthobium rubrum, A. gillii, and its recent segregate species, A. nigrum. The association of A. gillii with A. nigrum is strongly supported by ITS analysis (96% bootstrap). Bootstrap support is 75% for the entire clade containing A. rubrum. Because isozyme characters also indicated a grouping of these three species, their phyletic affinity is highly probable.

 

Arceuthobium pusillum and A. bicarinatum

A surprising but strongly supported clade (100% bootstrap) contains Arceuthobium pusillum and A. bicarinatum. The former species is a reduced parasite of spruce of the northern United States and Canada, and the latter is a relatively large parasite of Pinus occidentalis on the island of Hispaniola. Hawksworth and Wiens (1972) placed A. pusillum and A. douglasii in section Minuta, a placement not supported by isozyme analysis (Nickrent 1986). They also suggested that A. bicarinatum arrived in Hispaniola via a Central American land bridge that was connected to Honduras during the late Tertiary Period (see chapter 5). This route seemed plausible given the morphological similarity between A. bicarinatum and A. hondurense. The distance from Hispaniola to the nearest extant population of A. pusillum is about 2,300 km; A. bicarinatum and A. hondurense are presently separated by about 1,100 km.

Given these biogeographical distributions and the DNA sequence results, an alternate hypothesis regarding these two mistletoes is suggested. Several plant species found at high elevations in Hispaniola are known to have close relatives in the eastern United States, such as Lyonia (Judd 1981) and Juniperus (Adams 1989). Hawksworth and Wiens (1972) suggested that Arceuthobium arrived in the New World in the early Tertiary Period via a Beringian land bridge. If one accepts that the late Tertiary microthermal vegetation was largely derived from the preceding flora of that area (Wolfe 1975), then the ancestor to A. bicarinatum and A. pusillum was likely already present in eastern North America in the early Tertiary Period. Arceuthobium pollen (likely A. pusillum) is known from as far south as Georgia from the Pleistocene Epoch (Watts 1975 and table 5.10), hence dwarf mistletoes have occupied the eastern United States in past geological times. Evidence that some Arceuthobium species such as A. oxycedri were already well differentiated by the Miocene Epoch (Stuchlik 1964) also suggests evolution of the genus in the early or mid-Tertiary Period.

Configuration of the Caribbean region with respect to North and Central America during the Tertiary Period is an area of active research. In the later Paleocene to mid-Eocene Epochs, the Greater Antilles collided with the Bahama Platform (Pindell and Barrett 1990) and connected Cuba to the North American Plate. By the Miocene Epoch, Hispaniola was contiguous with the eastern part of Cuba, and northeastward displacement was occurring along the Oriental Fault. The connection between Honduras, Jamaica, and the Greater Antilles via the Nicaraguan Rise was likely severed during the middle Cenozoic Era by subsidence. This route to Hispaniola via Central America was proposed by Rosen (1975) as a "vicariance" pathway. But given the molecular evidence, entry into Hispaniola via eastern North America and Cuba is favored over the southwest track via Honduras.

The present lack of parasitism of low-elevation pines in Honduras, Belize, Cuba, Hispaniola, and the southeast United States indicates that an ancestral species was either already adapted to high-elevation hosts or that, subsequent to speciation, the low-elevation parasites became extinct. Even if Arceuthobium bicarinatum were found to be genetically similar to A. hondurense, this relationship would not favor one migration track over the other. These species would simply be a relictual taxon derived from the originally widespread ancestor. Given the overall climatic deterioration and the accompanying extinctions that occurred in eastern North America during the Oligocene Epoch (Tiffney 1985), this ancestral mistletoe is likely now extinct. The isolated position of A. bicarinatum on high-elevation pines of Hispaniola suggests that it is indeed a Tertiary relict.

Morphological differences between Arceuthobium pusillum and A. bicarinatum contrast with their high level of genetic similarity. High genetic variability as measured by isozymes and increased substitution rate at the nuclear ribosomal cistron suggests that A. pusillum is more genotypically variable than is outwardly apparent from its morphology. Sufficient diversity of pathogenicity genes apparently exists to allow this species to parasitize Larix laricina, Pinus strobus, P. resinosa, and P. banksiana, in addition to the spruces Picea mariana, P. glauca, and P. rubens, its three principal hosts. It is hypothesized that A. pusillum and A. bicarinatum represent morphologically divergent endpoints of lineages that have been shaped by quite different evolutionary forces.

The above genetic data on the modern species suggests that their ancestor likely possessed a large store of potential genetic variation that became manifest following diversifying selection. Increased fitness was attained by separate populations exploiting different environments and hosts. The reduction in shoot height, systemic broom formation, spring flowering, and rapid fruit maturation seen in Arceuthobium pusillum may represent adaptations to greater winter extremes, as occurred in other eastern North American plant species.

 

Subgenus Arceuthobium

Two of the most striking results of this study of ITS variation are the extreme divergence of Arceuthobium abietis-religiosae and A. oxycedri from the other taxa sampled and the similarity of these species to each other. These results require a modification of concepts regarding relationships among the three New World members of subgenus Arceuthobium as well as their relationship to Old World members of this subgenus.

Arceuthobium americanum and A. verticilliflorum are more closely related to each other than to any other species, as shown by 100% bootstrap confidence for their clade. This clade has more affinity with subgenus Vaginata than with A. abietis-religiosae and A. oxycedri—indicating a major divergence in the verticillately branched group during their evolution in the New World. Subgenus Vaginata was apparently derived from an ancestor shared with A. americanum and A. verticilliflorum. The extreme divergence of A. abietis-religiosae and A. oxycedri from the remaining species also could be interpreted as evidence of separate migrations into the New World. Further molecular work would greatly benefit from inclusion of additional Old World species, such as A. azoricum, A. chinense, A. juniperi-procerae, A. minutissimum, A. pini, and A. tibetense.

 

Intergeneric Relationships in the Viscaceae

Nickrent and Franchina (1990) published the first phylogenetic analysis of parasitic angiosperms using small-subunit 18S rRNA sequences. They showed that Phoradendron serotinum and Dendrophthora domingensis were apparently derived from the Santalaceae (represented by a sequence of Buckleya). Since that time, a large number of complete 18S rDNA sequences have been determined for representatives of all families of the Santalales (Nickrent 1992 and unpublished data). Over 1,000 angiosperm rbcL (ribulose bisphosphate carboxylase/oxygenase, large subunit) sequences exist, and their analysis has allowed unprecedented insight into the phylogeny of higher plants (Chase and others 1993).

Sequences of nuclear 18S rDNA and chloroplast rbcL genes representing all Viscaceae genera were determined to test the effect of phylogeny estimation given genes derived from different subcellular compartments and to allow integration into the immense rbcL database. Sequences used in this analysis were determined by direct sequencing of products generated via PCR, as discussed above for ITS sequencing. All rbcL sequencing primers were supplied by G. Zurawski (DNAX Research Institute, Palo Alto, CA). The sequence of Phoradendron serotinum was determined by Morgan and Soltis (1993). Discussion of these analyses provides insight into the phylogenetic position of Arceuthobium within the family.

 

18S rDNA and rbcL Sequence Analysis

The 18S rDNA sequences are approximately 1,805 bp in length and can be aligned manually given information on secondary structural features (Neefs and others 1990, Nickrent and Sargent 1991). On average, 1 to 5% of the sites are variable when comparisons are made among most flowering plants; higher rates of nucleotide substitution are observed in the parasitic angiosperm families Rafflesiaceae, Hydnoraceae, Balanophoraceae, and Viscaceae (Nickrent and Starr 1994). Given this higher evolutionary rate, parsimony analysis using PAUP (Swofford 1993) was employed to study relationships among Arceuthobium oxycedri, A. pendens, A. verticilliflorum, Dendrophthora clavata, D. domingensis, Ginalloa arnotiana, Korthalsella complanata, K. lindsayii, Notothixos leiophyllus, N. subaureus, Phoradendron californicum, P. serotinum, Viscum album, and V. articulatum (see "Species Examined" for host and collection data). In addition, the sequence of Antidaphne viscoidea (Eremolepidaceae) and Santalum album were used as outgroups. These species have been shown via global analysis of representatives of the entire order to be outside the Viscaceae.

For the 18S rDNA data, parsimony analysis using the branch and bound method yielded a tree of length 627. Five clades are present (fig. 15.7A, parts A to E). Clade A consists of the 3 Arceuthobium species; clade B, the 2 Notothixos species; clade C, Korthalsella and Ginalloa; clade D, Dendrophthora and Phoradendron; and clade E, the 2 Viscum species.

The relationship among Arceuthobium, Notothixos, and Viscum is unresolved. But with both rbcL and 18S rDNA sequences and high bootstrap values, Korthalsella forms a clade with Ginalloa and Dendrophthora forms a clade with Phoradendron.

Although components of these clades are stable, their relative topology is not. For example, all possible rearrangements of these clades add only one step to the tree, hence the topology is essentially a polytomy. This result is confirmed using bootstrap analysis.

The rbcL gene is consistently 1,428 bp in length (476 amino acids), hence manual alignment of the sequences is straightforward. The mean percentage of sites that differ between Santalum (the outgroup) and the 7 species of Viscaceae is 5.85% (from 5.39% for Korthalsella to 7.3% for Arceuthobium). The accelerated evolutionary rate of Arceuthobium is seen by its having over twice as many substitutions as Antidaphne when compared to the outgroup. Figure 15.7B is the strict consensus of 2 trees of length 305 bp with bootstrap confidences indicated at the nodes. Although fewer species were sequenced for the rbcL gene than with 18S rDNA, several relationships are concordant. The relationship among Arceuthobium, Notothixos, and Viscum is unresolved; but with both rbcL and 18S rDNA sequences, Korthalsella forms a clade with Ginalloa and Dendrophthora a clade with Phoradendron, both with high bootstrap values. Less support exists for the positions of these major groupings as indicated by the bootstrap value of 65 and 69%. Greater resolution could possibly occur with continued sampling of Arceuthobium, Notothixos, and Viscum, but rbcL may not provide sufficient characters to distinguish genera within the Viscaceae. Alternative faster-rate molecules should be examined to resolve relationships among these genera.

Taken together, results of the 18S rDNA and rbcL analyses support the concept that Phoradendron and Dendrophthora are closely related and that Viscum is likely the most primitive member of the genus (Wiens and Barlow 1971). It does not appear, however, that Korthalsella is the sister taxon to Arceuthobium nor that Ginalloa and Notothixos are closely related. The topology of the phylogenetic tree suggested by Wiens and Barlow (1971) is as follows: (((Ginalloa, Notothixos) Viscum) ((Korthalsella, Arceuthobium) (Phoradendron, Dendrophthora))). This arrangement of genera, however, results in an 18S rDNA tree 29 steps longer than the minimum length, hence it is unlikely that this topology best represents the phylogeny. Chromosome size and number may not be as useful for inferring phylogenetic relationships in Viscaceae as in Loranthaceae.

The evidence for linking Korthalsella with Arceuthobium has been the reported presence of "mildly explosive" fruits in Korthalsella. However, these fruits may either not really be explosive (Molvray, personal communication; Nickrent, unpublished data) or may represent another case of convergence. The molecular analyses did not indicate which genus is the closest relative of Arceuthobium. Biogeographic evidence suggests Notothixos or Viscum, because both have centers of diversity (and presumably centers of origin) in Asia, as was also postulated for Arceuthobium (Hawksworth and Wiens 1972).

Wiens (personal communication) questioned whether Arceuthobium verticilliflorum should be segregated as a separate genus from Arceuthobium given its unusually large and non-explosive fruits. The 18S rDNA and ITS sequence data clearly indicate that this species is properly placed in Arceuthobium. The loss of explosive seed dehiscence therefore represents an evolutionary reversal.

 

Summary of Molecular Evidence on Phylogenetic Relationships

Morphology, life cycles, and molecular data must be integrated to best estimate the phylogeny of Arceuthobium species. The following conclusions can be synthesized from existing information.

The genus of Viscaceae most closely related to Arceuthobium remains unresolved despite analyses of 18S rDNA and rbcL sequences. Previous phyletic hypotheses that derived Arceuthobium from Korthalsella are not supported by molecular evidence. Division of the genus into two subgenera (Arceuthobium and Vaginata) is supported by all analyses.

From ITS sequence data, the New World members of subgenus Arceuthobium comprise two groups (fig. 15.6). The New World Arceuthobium abietis-religiosae is genetically very similar to the Old World A. oxycedri and included with A. juniperi-procerae in section Arceuthobium (table 15.2). Arceuthobium abietis-religiosae is genetically distinct from other New World members of the subgenus Arceuthobium (A. americanum and A. verticilliflorum) that comprise the newly proposed section Americana (table 15.2). This high amount of genetic differentiation is also seen between A. oxycedri and both A. verticilliflorum and A. pendens when the more conservative 18S rDNA sequences are compared. Further sampling of ITS variation in other Old World members of subgenus Arceuthobium is needed.

The reconstituted subgenus Vaginata, section Vaginata (table 15.2), includes taxa previously in section Vaginata (Arceuthobium aureum, A. durangense, A. gillii, A. globosum, A. hawksworthii, A. nigrum, A. vaginata, and A. yecorense) as well as several species previously placed in section Campylopoda (A. divaricatum, A. oaxacanum, A. rubrum, and A. strictum) and in section Minuta (A. douglasii).

Neither isozyme nor DNA analyses cluster Arceuthobium douglasii with A. pusillum, but the association of A. douglasii with A. divaricatum receives strong support from isozyme data and moderate support from ITS-1 sequence data. Section Minuta should be reconstituted as series Minuta composed of A. douglasii and A. divaricatum (table 15.2).

Arceuthobium pusillum is genetically very similar to A. bicarinatum but has developed a large number of morphological and physiological apomorphies such as reduced shoot size and systemic broom formation. These two species and A. hondurense are the likely survivors of an ancient lineage whose most recent common ancestor became extinct during the Tertiary Period. These species are recognized as members of section Pusilla (table 15.2).

Two additional segregates, Arceuthobium pendens and A. guatemalense, from the former section Campylopoda are deserving of placement in the new section Penda. These species are narrow endemics and are also likely relicts of a common Tertiary ancestor.

The remaining section Campylopoda now comprises 13 species mainly of the United States and with a high degree of morphological and genetic similarity. The reconstituted section includes Arceuthobium abietinum, A. apachecum, A. blumeri, A. californicum, A. campylopodum, A. cyanocarpum, A. laricis, A. littorum, A. microcarpum, A. monticola, A. occidentale, A. siskiyouense, and A. tsugense.

The observations made by Sytsma and Smith (1992) regarding concordance or discordance between morphological and molecular divergence in Clarkia and Fuchsia are applicable to dwarf mistletoes. Basically, four syndromes were described: low morphological and molecular divergence, high morphological and high DNA divergence, low morphological but high DNA divergence, and high morphological but low DNA divergence. Clarkia ligulata and C. biloba are given as examples for the first syndrome. In Arceuthobium, a similar situation occurs among members of section Campylopoda. The second syndrome was illustrated by comparing sections Myxocarpa and Eucharidum of Clarkia or the Old World section Skinnera with New World sections. In Arceuthobium, the most morphological divergence is seen between representatives of subgenera Arceuthobium and Vaginata. This distinction is strongly supported by molecular evidence. The third syndrome can be demonstrated by comparing C. rostrata with C. lewisii and C. cylindrica. In dwarf mistletoes, a low amount of morphological divergence is seen between A. pendens and A. guatemalense as compared with species in section Campylopoda (however, DNA evidence indicates these two groups are not closely related). Finally, the fourth syndrome can be seen in comparisons of Clarkia rostrata and C. epilobioides which differ in breeding system and floral morphology but are extremely similar genetically. Similarly, Heterogaura heterandra and C. dudleyana exhibit this type of discordance. For Arceuthobium, this syndrome is best shown by A. pusillum and A. bicarinatum or by A. douglasii and A. divaricatum.

The dwarf mistletoes continue to present a challenge to the systematist, as do all parasitic plants that follow reductional and/or convergent evolutionary paths. These plants provide the ultimate test of our abilities to reconstruct phylogenies, therefore alternate data sets for other genes must be assembled to confirm or support proposed relationships.

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