Inheritance patterns of ITS1, chloroplasts and mitochondria in artificial ...

Eur. J. Phycol. (2002), 37 : 173–178. # 2002 British Phycological Society DOI : 10.1017\S0967026202003682 Printed in the United Kingdom

173

Inheritance patterns of ITS1, chloroplasts and mitochondria in artificial hybrids of the seaweeds Fucus serratus and F. evanescens (Phaeophyceae)

J . A . C O Y E R1 , A . F . P E T E R S2 , G . H O A R A U1 , W . T . S T A M1 A N D J . L . O L S E N1 " Department of Marine Biology, Centre for Ecological and Evolutionary Studies, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands # Marine Oq kologie, Institut fuW r Meereskunde, DuW sternbrooker Weg 20, 24105 Kiel, Germany (Received 2 October 2001 ; accepted 7 February 2002) Patterns of nuclear and organelle inheritance among artificial hybrids of the seaweeds Fucus serratus and F. evanescens were detected using single-strand conformation polymorphism (SSCP). Three alleles were identified in the 231 bp rDNA-ITS1 gene (nuclear) : two in F. serratus and one in F. evanescens. Alleles differed by 1–2 bp and all hybrids possessed one allele from each parent. Two haplotypes were present in the 288 bp Rubisco spacer (chloroplast), differentiated by a 33 bp indel. Two haplotypes differing by a single nucleotide were found in a 135 bp region of nad11 gene (mitochondrion). Both organelles are maternally inherited, as all hybrids contained the haplotypes of the parent contributing the egg. Although laboratory hybrids among Fucus spp. have been produced previously, this is the first time that both nuclear and cytoplasmic genetic markers have been used to document inheritance patterns. SSCPs analysed on an automated sequencer offer a rapid and powerful approach for identifying suspected hybrids from field samples, as well as a screen for intraspecific and intra-individual variation in DNA regions prior to confirmation of variations by sequencing. Key words : Fucus serratus, F. evanescens, hybrids, ITS1, nad11, organelle inheritance, Rubisco spacer, SSCP

Introduction Analysis of the DNA sequence in nuclear and organellar genes and spacers of seaweeds has been widely applied at the phylogenetic (Olsen et al., 1998 ; Draisma et al., 2001) and biogeographical (Peters et al., 1997 ; Meusnier et al., 2001) scales, as well as at the population level (Coyer et al., 2001). Central to their importance as genetic markers, however, is an understanding of their mode of transmission from parent to offspring. For example, transmission of animal mitochondria is through maternal lines in most species, although several exceptions exist and, in some species, such as marine mussels, ‘ paternal leakage ’ is relatively common (reviewed in Avise, 1994). In plants, mitochondrial inheritance is usually maternal, whereas chloroplast inheritance can be maternal (usually), biparental or paternal (reviewed in Avise, 1994 ; Morgensen, 1996 ; see also Isoda et al., 2000). Little is known about inheritance patterns of nuclear and organellar sequences in marine macroalgae. Only one study has documented inheritance patterns of internal transcribed spacer (ITS) regions in laboratory crosses using molecular markers Correspondence to : J. Coyer. e-mail : coyerja!biol.rug.nl

(Liptack & Druehl, 2000). Chloroplast inheritance has been examined in four species using electron microscopy (Bouck, 1970 ; Bisalputra et al., 1971 ; Brawley et al., 1976 a ; Motomura, 1990) and only one species using genetic markers (Zuccarello et al., 1999 a). Mitochondria are less commonly used for phylogeny or population studies in macroalgae and inheritance patterns have been investigated with electron microscopy in two species (Brawley et al., 1976 b ; Motomura, 1990) and with molecular markers in one (Zuccarello et al., 1999 b). In marine macroalgae, verification of hybrid status for individuals with morphologies intermediate between two co-occurring species currently depends upon crossing experiments, which are difficult, labour-intensive and often not feasible. In the Kattegat and Baltic Sea, populations of Fucus serratus L. and F. evanescens C. Ag. (Phaeophyceae) have become sympatric since F. evanescens was introduced to the Oslofjord in the 1890s (Schueller & Peters, 1994). Putative hybrids of the two species were observed in Oslofjord in the late 1970s (Lein, 1984 ; Rice & Chapman, 1985) and from 1998 to 2000 wherever the two species co-occurred in the Kattegat to western Baltic Sea (Coyer and Peters, unpublished data). Therefore, it was of interest to

J. A. Coyer et al. develop appropriate genetic markers that could be used to rapidly confirm the existence of hybrids and hybridization in field populations. The aims of the present study were to : (1) verify hybridization in laboratory crosses of F. serratus and F. evanescens using genetic markers and single-strand conformational polymorphism (SSCP), and (2) assess the inheritance patterns of nuclear and organellar markers in Fucus. Materials and methods Artificial hybrids were produced in the laboratory using fertile parental individuals collected from Blushøj (near Elsega/ rde), Denmark (56m 10h N, 10m 43h E) in April 2000. Individual receptacles were placed in separate plastic bags and transported to the laboratory on ice. The sex of individual Fucus serratus was determined microscopically. In the dioecious F. serratus, all receptacles of an individual contain either antheridia or oogonia. Receptacles from mature F. serratus thalli were stored in plastic bags overnight at 0–4 mC. The addition of ice-cold sterilized seawater induced conceptacles to release antheridia or oogonia, which were collected and washed once in sterile seawater before further use. As conceptacles of F. evanescens contain both antheridia and oogonia (l hermaphroditic), collecting oogonia after release from conceptacles was inappropriate, because eggs from such preparations already were fertilized by conspecific sperm. Hence, to prevent selfing in F. evanescens, oogonia were collected from conceptacles immediately after sectioning them with a razor blade, and washed (i3) in sterile seawater before use. Clusters of F. evanescens antheridia were obtained in the same manner. Oogonia of F. serratus or F. evanescens isolated as described above were inoculated with or without antheridia. A reciprocal crossing design was used (Table 1) in which F. evanescens females were crossed with F. serratus males and males of the same F. evanescens individuals were crossed with F. serratus females. A set of crosses consisted of these two combinations, plus negative controls consisting of eggs only (no sperm added) and positive controls consisting of conspecific crosses. Both negative and positive controls were performed for each of the species involved. Crosses were performed in sterile plastic dishes containing 3 ml sterile seawater at 5 mC. We made nine different sets of crosses by combining nine individuals of F. evanescens with nine male and nine female individuals of F. serratus. To allow quantification of fertilization success for an unpublished study, each cross and control was replicated four times using 10 oogonia in each replicate. Replication also served to increase the probability of obtaining isolates not contaminated by small filamentous algae, because unialgal cultures were required for subsequent raising of thalli to the size necessary for DNA extraction. After inoculation, dishes were incubated at 20 µmol m−# s−" white light at 5 mC. Three days after the start of the experiments, the seawater was replaced by culture medium (half-strength Provasoli’s Enriched Seawater ; Provasoli, 1963) containing 6 mg l−" GeO to prevent growth of diatoms. Replenishment of # culture medium occurred at 14 day intervals, but without GeO . #

174 Table 1. Reciprocal crossings between Fucus serratus (Fs) and F. evanescens (Fe)

Cross

Rubisco ITS1 spacer nad11 (nuclear) (chloroplasts) (mitochondria)

FeL (2081)iFsK (5000) L19 L20 L21

αiβ αβ αβ αβ

AiB A A A

1i2 1 1 1

FeL (2087)iFsK (5009) L58 L60 L62

αiβ αβ αβ αβ

AiB A A A

1i2 1 1 1

FeL (2094)iFsK (5016) L45 L46 L48

αiβ αβ αβ αβ

AiB A A A

1i2 1 1 1

FeL (2095)iFsK (5017) L35 L36 L37 Fs L (5003)iFeK (2081) L24 L25 L26

αiβ αβ αβ αβ γiα γα γα γα

AiB A A A BiA B B B

1i2 1 1 1 2i1 2 2 2

Fs L (5018)iFeK (2095) L38 L41 L42

βiα αβ αβ αβ

BiA B B B

2i1 2 2 2

Fs L (5019)iFeK (2094) L50 L51 L52

βiα αβ αβ αβ

BiA B B B

2i1 2 2 2

Fs L (5023)iFeK (2087) L63 L64 L65

βiα αβ αβ αβ

BiA B B B

2i1 2 2 2

FeLiFeK (2081C) FsL (5023C)iFsK (5009C)

α β

A B

1 2

Results are shown from four different crossing experiments involving four parent individuals of F. evanescens and eight of F. serratus. Three F1 thalli from each cross are presented. Individuals of F. evanescens served as females (upper half of table) or males (lower half ) because they are hermaphroditic. Values in brackets are individual-specific identification codes and a subscript c indicates positive control crosses for each species. Alleles and haplotypes derived from portions of ITS1 (nuclear), Rubisco (chloroplast) and nad11 (mitochondrial) genes as determined by SSCP. GenBank accession numbers for sequences and alignments : α (AY044258), β (AY044260), γ (AY044259), A (AY044263), B (AY044264), 1 (AY044261) and 2 (AY044262).

After 4 weeks, embryos were identified by their typical club-shape, the presence of an apical tip with hair pit, and growth of basal rhizoids. Of the nine original sets of crosses, two were discarded at this stage because embryos were present in the negative controls of F. evanescens, which indicated selfing. Only unialgal cultures (nonaerated) were maintained, in which thalli reached 5– 50 mm in length after 7–11 months at 10–15 mC. Offspring of four sets of crosses were finally harvested for DNA (using 5–20 mg of fresh tissue) as previously described (Coyer et al., 2002).

40

2

F. serratus (Oudot-Le Secq, unpublished. data)

175

See Table 1 legend for GenBank accession numbers of primers.

135 F : 5h-TTTGGTAGAGGTAGGTAACG (FAM) R : 5h-TGTAACAGAAGTAATTCCATA (NED)

42 255, 288

2

F. evanescens (AF102939) F. serratus (AF102943) F. distichus (AF195515) F. vesiculosus (AF132474) 3 47 231

F : 5h-TCGACCAAACGTGTCTGTTT (FAM) R : 5h-ACGCTAGGCTTCCTTCCTTC (NED) F : 5h-TGAATATACTTCAACAGATACACC (FAM) R : 5h-TGGTAAAAATGAAAAACATCCTTG (NED)

rDNA-ITS1 (nuclear) Rubisco spacer with rbcL and rbcS flanks (chloroplast) nad11 (mitochondrion)

Primers (fluorescent label) Gene

Table 2. Identity and characteristics of primers used for SSCP analysis

Fragment size (bp)

Ta (mC)

No. of alleles\haplotypes

Reference sequences for primer design

ITS and organelle inheritance in Fucus

Sequence polymorphisms were revealed by singlestrand conformation polymorphism (SSCP ; Orita et al., 1989 ; Hayashi, 1992). The technique detects sequence variation by the differential migration of single-stranded DNA in non-denaturing, high-resolution, polyacrylamide gels. Using SSCP, differences of one or more nucleotides can be detected in fragments from 300 to 450 bp with nearly 90 % accuracy (Hayashi, 1991 ; Lessa & Applebaum, 1993). As mutations may affect the mobility of only one or both of the two DNA strands (Hoarau et al., 1999 ; Lescasse, 1999), labelling each strand independently confers additional sensitivity. Independent labelling was achieved by labelling the forward and reverse primers (Table 2) with a different fluorescent dye and using an ABI 377 autosequencer (Applied Biosystems) for detection. SSCP allows a rapid and inexpensive means to determine sequence differences (as revealed by conformational differences) for a large number of samples (Sunnucks et al., 2000). SSCP alleles\haplotypes were detected by polymerase chain reaction (PCR) amplification of the ITS1 (nuclear), ribulose-1,5-bisphosphate carboxylase operon (Rubisco ; chloroplast), and nad11 (NADH dehydrogenase, subunit 11 ; mitochondrion) (Table 2). A portion of the Fucus nad11 gene was amplified using PCR primers designed from the nad11 sequence determined for Pylaiella littoralis (L.) Kjellm (Ectocarpales) (Oudot-Le Secq et al., 2001). Although microsatellite loci (nuclear) have been developed for F. serratus and F. evanescens (Coyer et al., 2002), their high levels of polymorphism would require numerous samples to discern parental\hybrid patterns. Consequently, the much less polymorphic ITS1 gene was used as the nuclear marker in the present study so that parental\hybrid patterns could be detected with fewer individuals. PCR reactions (10 µl total volume) contained 1 µl of the DNA extract (Coyer et al., 2002), 1iTaq polymerase buffer (Promega), 2 mM MgCl , 0n2 mM of each dNTP, # 0n1 µM (ITS1, nad11) or 0n4 µM (Rubisco) of each primer (one of each primer pair 5h-labelled with FAM, the other with NED ; Applied Biosystems), and 0n25 or 0n50 U Taq polymerase (Promega). Additionally, bovine serum albumin (BSA ; 0n8 µg µl−") was added to PCR reactions for Rubisco and nad11. PCR was performed with a Mastercycler Gradient (Eppendorf ) thermocycler (94 mC, 3 min ; followed by 94 mC, 40 s ; annealing temperature (Ta), 40 s ; and 72 mC, 40 s for 40 cycles (42 cycles for Rubisco) ; and a final extension at 72 mC for 10 min). Amplification products were separated in a 0n4iMDE gel (BMA Bioproducts) in 0n6 % TBE at 19 mC and 45 W on an ABI 377 autosequencer. Samples (0n5–1n0 µl) were loaded with 1n5 µl of loading buffer containing 67 % deionized formamide, 11 % GeneScan size standard ROX 350 (Applied Biosystems), 11 % Blue dextran EDTA (Applied Biosystems) and 11 mM NaOH. Alleles or haplotypes were identified with GeneScan software (Applied Biosystems). All unique SSCP polymorphisms for each gene were verified by direct sequencing of the PCR product (both strands) from three individuals using the dGTP BigDye Terminator Kit and ABI 377 autosequencer (Applied Biosystems). As the sequences of the SSCP polymorphism were identical in each of the three individuals, the SSCP polymorphisms correspond to alleles (ITS) or haplotypes (chloroplast, mitochondrion). Only one of the three

J. A. Coyer et al. identical sequences for each allele\haplotype was placed into GenBank (Table 1).

Results Both sets of reciprocal crosses produced hybrid individuals, and segregation of alleles or haplotypes for each gene was clear and unambiguous (Fig. 1). Three alleles were identified for the 231 bp ITS1 region. Fucus serratus displayed alleles β and γ, whereas F. evanescens possessed only allele α (Table 1). Subsequent sequencing and alignment (retrievable from GenBank accession numbers in Table 1) of the three alleles revealed a difference between α and β at position 87 (A\C) and α and γ at position 199 (G\A). All laboratory hybrids possessed two alleles, one from each parent. Two haplotypes were identified for the Rubisco spacer (chloroplast). Fucus serratus displayed haplotype A (255 bp) and F. evanescens haplotype B (288 bp) (Table 1). Subsequent sequencing of the

176 two fragments revealed a 33 bp deletion (positions 73–106). The hybrids possessed only one haplotype, which matched the female parent in all cases. Two haplotypes also were identified for a 135 bp region of nad11 (mitochondrion) (Table 1). Sequencing of three individuals with haplotype 1 (F. evanescens) and three individuals with haplotype 2 (F. serratus) revealed a single nucleotide difference at position 85 (T\C) in the alignment. Once again, the hybrids contained only one haplotype, which always corresponded to the female parent. It is highly unlikely that the single-base difference observed in the mitochondrial haplotypes was due to errors in PCR amplification. First, each haplotype was sequenced from three separate individuals and, as the three sequences were identical, either there were no PCR errors or errors occurred at the same base in each of three separate reactions (highly unlikely). Secondly, only two SSCP patterns (which are dependent upon sequence) were present among all 24 F1 thalli. If PCR errors were apparent in our SSCP analysis, it is improbable that such errors would occur at the same base in each of the 24 separate reactions.

Discussion

Fig. 1. Hybrid inheritance patterns based on singlestrand conformation polymorphism (SSCP). Each strand was labelled independently with FAM (grey peaks) or NED (black peaks) and visualized on an ABI 377 autosequencer. Patterns presented are representative of all samples. (A) ITS1 (nuclear) ; (B) Rubisco (chloroplast) ; (C ) nad11 (mitochondrion). Abbreviations : Fs, Fucus serratus ; Fe, F. evanescens ; numbers refer to sample codes.

Identification of interspecific hybrids and subsequent study of hybrid zones are of great importance in understanding speciation and the effects of species introductions on extant communities ; nevertheless, the degree and extent of hybridization in marine macroalgae are essentially unknown. As a prelude to such studies, we have documented patterns of gene inheritance in laboratory hybrids of the seaweeds Fucus serratus and F. evanescens, using crossing experiments and three genetic markers. The ITS1 region in F. serratus and F. evanescens is biparentally inherited, as expected from a nuclear gene. All laboratory hybrids possessed one allele from each parent. The point mutations in the F. serratus ITS1 sequences, which formed the basis for the SSCP polymorphism, were consistent in all of our F. serratusiF. evanescens crosses, strongly suggesting that the ITS1 polymorphism is stable and will be useful in identifying hybrids from the field. Microsatellite loci (nuclear) should prove useful for reliable distinction of second-generation hybrids (F2), backcrosses to either of the parental taxa, and later-generation hybrids (Allendorf et al., 2001). It must also be recognized that the ITS1 is part of a multi-copy, multi-gene family in which internal homogeneity of the ITS repeat units is dependent on concerted evolution (Hillis & Davis, 1988 ; Williams et al., 1988). Intraspecific and intra-individual polymorphism of ITS is well documented in marine

ITS and organelle inheritance in Fucus seaweeds (Pillmann et al., 1997 ; Serra4 o et al., 1999 ; Fama' et al., 2000 ; Coyer et al., 2001), presumably related to incomplete homogenization under concerted evolution (Dover, 1982) as a result of recent speciation, hybridization, asexual reproduction, polyploidy or multi-chromosomal locations. In F. vesiculosus, for example, the 1–2 % difference observed in ITS1 and ITS2 sequences within a single individual was attributed to frequent hybridization and\or a rate of radiation exceeding the homogenization rate by concerted evolution (Serra4 o et al., 1999). Our finding of two ITS1 alleles among only eight individuals of F. serratus illustrates that polymorphism can occur among individuals separated by a few metres, a degree of intraspecific ITS polymorphism also found in the kelp, Macrocystis pyrifera (Coyer et al., 2001). Although we found no evidence for intra-individual polymorphism in Fucus (i.e. multiple peaks in SSCP analysis), such a result cannot be eliminated, especially if sampling intensity were increased and\or naturally occurring F. serratusiF. evanescens hybrids were reproductive and backcrosses were prevalent. Chloroplasts and mitochondria are maternally inherited in F. serratus and F. evanescens. In all cases (Table 1), the organellar haplotype found in the hybrid was identical to that present in the eggcontributing parent. With respect to chloroplasts, our SSCP-based results obtained from severalmonth old sporophytes differed from previous electron-microscopy-based studies. Both Bouck (1970) and Brawley et al. (1976 a) observed sperm chloroplasts in embryos of F. vesiculosus, which suggested paternal inheritance. The discrepancy most likely results from comparing different developmental stages in which older individuals have lost the traces of paternal chloroplasts. In the kelps, electron microscopy suggested paternal and maternal inheritance of chloroplasts in Laminaria ephemera and L. saccharina (Bisalputra et al., 1971), but essentially maternal inheritance in L. angustata (Motomura, 1990). Again, the equivocal nature of the electron microscopy data may be a result of using different developmental stages for analysis. In the only other study that has investigated chloroplast inheritance in marine macroalgae, Zuccarello et al. (1999 a) used SSCP with short fragments of the Rubisco spacer on mature individuals of the red alga Bostrychia (Ceramiales) and demonstrated maternal inheritance of chloroplasts. Our results support earlier studies investigating mitochondrial inheritance in three species of marine macroalgae. Degenerated sperm mitochondria were observed in 16 h embryos of F. vesiculosus (Brawley et al., 1976 b) and in pre-division zygotes of the kelp Laminaria angustata (Motomura, 1990) ; both results were interpreted as evidence for maternal inheritance. Maternal inheritance of mitochondria

177 was also demonstrated in the red alga Bostrychia moritziana using sequence variability in the noncoding spacer between the cytochrome oxidase subunits 2 and 3 genes (Zuccarello et al., 1999 b). The universality of maternal inheritance of organelles in marine macroalgae remains to be determined. Given the phyletic diversity of macroalgae, however, it is probable that both maternal and paternal inheritance patterns will be found. SSCPs analysed on an automated sequencer offer a rapid and powerful approach for surveying inheritance patterns and polymorphisms in a large number of samples. For example, selection of samples for subsequent sequencing can be made from preliminary SSCP analysis, thereby identifying polymorphisms that would be missed by direct sequencing unless expensive and time-consuming cloning techniques were employed. SSCP analysis has also verified that our method of producing artificial hybrids is reliable even in hermaphroditic taxa such as F. evanescens. Notably, it was not necessary to immobilize F. evanescens sperm by the addition of reagents, such as potassium salts, sodium hypochlorite or methanol (Fritsch, 1945), which might have affected viability of the eggs as well. Finally, SSCP can identify field samples putatively identified as F. evanescensiF. serratus hybrids, provided that examination of an adequate number of parental samples reveals species-specificity of the markers and no intraspecific heterogeneity. Subsequent tests of hybrid fitness and viability in relation to hybrid zones and dispersal will provide new insights into community structure and the evolution of seaweeds.

Acknowledgements We thank M.-P. Oudot-Le Secq for providing unpublished sequences of mitochondria from F. serratus and R. Lescasse for technical assistance in using autosequencers for SSCP analysis. The research was supported by the BIOBASE Project funded under EU MAST III, Control Number PL97-1267.

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